Human tumor necrosis factor receptors TR21 and TR22

ABSTRACT

The present invention relates to two novel proteins, TR21 and TR22, which are members of the tumor necrosis factor (TNF) receptor. In particular, isolated nucleic acid molecules are provided encoding the human TR21 and TR22 protein. TR21 and TR22 polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of TR21 and TR22 activity; and methods of treating immune disorders by administering TR21 and TR22 polynucleotides, polypeptides, agonists, and antagonists.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims benefit under 35 U.S.C.§ 120 of non provisional Application No. 09/910,562, filed on Jul. 23,2001, which in turn claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/220,116, filed Jul. 24,2000, and 60/221,143, filed Jul. 27, 2000, each of which non provisionaland provisional applications is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to a novel member of the tumor necrosisfactor family of receptors. More specifically, isolated nucleic acidmolecules are provided encoding a novel human tumor necrosis factorreceptor, TR21 and TR22. TR21 and TR22 polypeptides are also provided,as are vectors, host cells, and recombinant methods for producing thesame, and antibodies and peptides that bind to TR21 and TR22polypeptides. The invention further relates to screening methods foridentifying agonists and antagonists of TR21 and TR22 activity.

BACKGROUND OF THE INVENTION

Many biological actions, for instance, response to certain stimuli andnatural biological processes, are controlled by factors, such ascytokines. Many cytokines act through receptors by engaging the receptorand producing an intra-cellular response.

For example, tumor necrosis factors (TNF) alpha and beta are cytokines,which act through TNF receptors to regulate numerous biologicalprocesses, including protection against infection and induction of shockand inflammatory disease. The TNF molecules belong to the “TNF-ligand”superfamily, and act together with their receptors or counter-ligands,the “TNF-receptor” superfamily. So far, nine members of the TNF ligandsuperfamily have been identified and ten members of the TNF-receptorsuperfamily have been characterized.

Among the ligands there are included TNF-, lymphotoxin- (LT- , alsoknown as TNF-β), LT-β (found in complex heterotrimer LT-2-β), FasL,CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF). Thesuperfamily of TNF receptors includes the p55TNF receptor, p75TNFreceptor, TNF receptor-related protein, FAS antigen or APO- 1, CD40,CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (A. Meager,Biologicals 22:291-295 (1994)).

Many members of the TNF-ligand superfamily are expressed by activatedT-cells, implying that they are necessary for T-cell interactions withother cell types which underlie cell ontogeny and functions. (A. Meager,supra).

Considerable insight into the essential functions of several members ofthe TNF receptor family has been gained from the identification andcreation of mutants that abolish the expression of these proteins. Forexample, naturally occurring mutations in the FAS antigen and its ligandcause lymphoproliferative disease (R. Watanabe-Fukunaga et al., Nature356:314 (1992)), perhaps reflecting a failure of programmed cell death.Mutations of the CD40 ligand cause an X-linked immunodeficiency statecharacterized by high levels of immunoglobulin M and low levels ofimmunoglobulin G in plasma, indicating faulty T-cell-dependent B-cellactivation (R. C. Allen et al., Science 259:990 (1993)). Targetedmutations of the low affinity nerve growth factor receptor cause adisorder characterized by faulty sensory innovation of peripheralstructures (K. F. Lee et al., Cell 69:737 (1992)).

TNF and LT- are capable of binding to two TNF receptors (the 55- and75-kd TNF receptors). A large number of biological effects elicited byTNF and LT-, acting through their receptors, include hemorrhagicnecrosis of transplanted tumors, cytotoxicity, a role in endotoxicshock, inflammation, immunoregulation, proliferation and anti-viralresponses, as well as protection against the deleterious effects ofionizing radiation. TNF and LT- are involved in the pathogenesis of awide range of diseases, including endotoxic shock, cerebral malaria,tumors, autoimmune disease, AIDS and graft-host rejection (B. Beutlerand C. Von Huffel, Science 264:667-668 (1994)). Mutations in the p55receptor cause increased susceptibility to microbial infection.

Moreover, an about 80 amino acid domain near the C-terminus of TNFR1(p55) and Fas was reported as the “death domain,” which is responsiblefor transducing signals for programmed cell death (Tartaglia et al.,Cell 74:845 (1993)).

Apoptosis, or programmed cell death, is a physiologic process essentialto the normal development and homeostasis of multicellular organisms (H.Steller, Science 267:1445-1449 (1995)). Derangements of apoptosiscontribute to the pathogenesis of several human diseases includingcancer, neurodegenerative disorders, and acquired immune deficiencysyndrome (C. B. Thompson, Science 267:1456-1462 (1995)). Recently, muchattention has focused on the signal transduction and biological functionof two cell surface death receptors, Fas/APO-1 and TNFR-1 (J. L.Cleveland et al., Cell 81:479-482 (1995); A. Fraser et al., Cell85:781-784 (1996); S. Nagata et al., Science 267:1449-56 (1995)). Bothare members of the TNF receptor family, which also include TNFR-2, lowaffinity NGFR, CD40, and CD30, among others (C. A. Smith et al., Science248: 1019-23 (1990); M. Tewari et al., in Modular Texts in Molecular andCell Biology M. Purton, Heldin, Carl, Ed. (Chapman and Hall, London,1995). While family members are defined by the presence of cysteine-richrepeats in their extracellular domains, Fas/APO-1 and TNFR-1 also sharea region of intracellular homology, appropriately designated the “deathdomain,” which is distantly related to the Drosophila suicide gene,reaper (P. Golstein et al., Cell 81:185-6 (1995); K. White et al.,Science 264:677-83 (1994)). This shared death domain suggests that bothreceptors interact with a related set of signal transducing moleculesthat, until recently, remained unidentified. Activation of Fas/APO-1recruits the death domain-containing adapter molecule FADD/MORTl (A. M.Chinnaiyan et al., Cell 81:505-512 (1995); M. P. Boldin et al., J. Biol.Chem. 270:7795-8 (1995); F. C. Kischkel et al., EMBO 14:5579-5588(1995)), which in turn binds and presumably activates FLICE/MACH1, amember of the ICE/CED-3 family of pro-apoptotic proteases (M. Muzio etal., Cell 85: 817-827 (1996); M. P. Boldin et al., Cell 85:803-815(1996)). While the central role of Fas/APO-1 is to trigger cell death,TNFR-1 can signal an array of diverse biological activities—many ofwhich stem from its ability to activate NF-kB (L. A. Tartaglia et al.,Immunol Today 13:151-153 (1992)). Accordingly, TNFR-1 recruits themultivalent adapter molecule TRADD, which like FADD, also contains adeath domain (H. Hsu et al., Cell 81:495-504 (1995); H. Hsu et al., Cell84:299-308 (1996)). Through its associations with a number of signalingmolecules including FADD, TRAF2, and RIP, TRADD can signal bothapoptosis and NF-kB activation(H. Hsu et al., Cell 84:299-308 (1996); H.Hsu et al., Immunity 4:387-396 (1996)).

Another apoptosis inducing TNF ligand was reported in S. R. Wiley etal., Immunity 3:673-682 (1995), which named the new molecule,“TNF-related apoptosis-inducing ligand” or “TRAIL.” R. M. Pitti et al.,J. Biol. Chem. 271:12687-12690 (1996), named the molecule “Apo-2 ligand”or “Apo-2L.” This molecule was also disclosed in InternationalPublication No. WO 97/33899. For convenience, this molecule will bereferred to herein as TRAIL.

Unlike FAS ligand, whose transcripts appear to be largely restricted tostimulated T-cells, significant levels of TRAIL are detected in manyhuman tissues (e.g., spleen, lung, prostate, thymus, ovary, smallintestine, colon, peripheral blood lymphocytes, placenta, kidney), andit is constitutively transcribed by some cell lines. It has been shownthat TRAIL acts independently from the FAS ligand (S. R. Wiley et al.,supra). It has also been shown that TRAIL activates apoptosis rapidly,within a time frame that is similar to death signaling by Fas/Apo-1L,but much faster than TNF-induced apoptosis. S. A. Marsters et al.,Current Biology 6:750-752 (1996). The inability of TRAIL to bind TNFR-1,Fas, or the recently identified DR3, suggests that TRAIL may interactwith a unique receptor(s).

Work to date suggests that there are several unique TNF receptors forTRAIL. International Publication No. WO 97/33904, one novel death domaincontaining receptor for TRAIL, DR4, was disclosed. See, Pan et al.,Science 276:111-113 (April 1997). In International Publication No. WO9841629, a novel death domain containing receptor, DR5 (TR7), wasdisclosed. This receptor has now been shown to bind TRAIL. InInternational Publication No. WO 98/30693, another receptor, TR5, wasdisclosed. This receptor has also now been shown to bind TRAIL, however,TR5 has been shown to be a non-signaling decoy receptor whichantagonizes apoptosis.

The effects of TNF family ligands and receptors are varied and influencenumerous functions, both normal and abnormal, in the biologicalprocesses of the mammalian system. There is a clear need, therefore, foridentification and characterization of such receptors and ligands thatinfluence biological activity, both normally and in disease states. Inparticular, there is a need to isolate and characterize additional novelreceptors that bind TRAIL.

SUMMARY OF THE INVENTION

TR21 and TR22 are novel receptor proteins that are highly homologous toeach other, and are members of the tumor necrosis factor (TNF) receptorfamily, with a high degree of homology to TR12 (InternationalPublication No. WO 00/23572). The present invention provides isolatednucleic acid molecules comprising or, alternatively, consisting of apolynucleotide encoding (a) the TR21 receptor having the amino acidsequence shown in FIGS. 1A-B, or the amino acid sequence encoded by cDNAclone number HCFMV39 contained in American Type Culture Collection(“ATCC”) Deposit No. 97974, deposited on Apr. 4, 1997, or contained inATCC Deposit No. 209080, deposited May 29, 1997; and (b) the TR22receptor having the amino acid sequence shown in FIG. 2, or the aminoacid sequence encoded by cDNA clone number HMUCL01 contained in ATCCDeposit No. PTA-2259, deposited on Jul. 25, 2000. The ATCC is located at10801 University Boulevard, Manassas, Va. 20110-2209.

The nucleotide and amino acid sequences for TR22 shown in FIG. 2 arepartial sequences. Nonetheless these sequences are sufficiently completeto identify the TR22 protein as a unique, novel member of the TNFreceptor family. Based on the sequence shown in FIG. 2 and the HMUCL01clone contained in ATCC Deposit No. PTA-2259, the skilled person canobtain the full length TR22 protein, the mature TR22 protein, as well asother embodiments of the TR22 protein that contain amino acids that arenative to the TR22 protein but not shown in FIG. 2. Indeed, suchadditional embodiments are inherently encompassed by the TR22 proteinencoded by the HMUCL01 clone of ATCC Deposit No. PTA-2259.

In specific embodiments related to TR21 receptor, polypeptides of theinvention comprise the following amino acid sequence:STHASGPPAPERLCLPERGTAP WGRRANDAA (SEQ ID NO: 5),VRRWWLRTMGAAAHCTPEQRRPRRPATILGMDTQNILHTRLSLCSLSWVSLASSFXXLAXRRKAIVVQQKQSKISKKKKVEKXXLNDSVNENSDTVGQIVHYIMKNEANADVLKAMVADNSLYDPESPVTPSTPGSPPVSPGLCHQGGRQGSTSVAIICIRWAVXSRGMCVIGVGTSGGTL (SEQ ID NO: 6), and/orIMKNEANADVLKAMVADNSLYDPESPVTP (SEQ ID NO: 7). Polynucleotides encodingthese polypeptides are also encompassed by the invention.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to the nucleotide sequences of FIGS. 1A-B and 2and may have been publicly available prior to conception of the presentinvention. Preferably, such related polynucleotides are specificallyexcluded from the scope of the present invention. To list every relatedsequence is cumbersome. Accordingly, preferably excluded from thepresent invention are one or more polynucleotides comprising anucleotide sequence described by the general formula of a−b, where a isany integer between 1 to 1275 of FIGS. 1A-B, b is an integer of 15 to1291, where both a and b correspond to the positions of nucleotideresidues shown in FIGS. 1A-B, and where b is greater than or equal toa+15; and by the general formula c−d, where c is any integer between 1to 566 of FIG. 2, b is an integer of 15 to 582, where both c and dcorrespond to the positions of nucleotide residues shown in FIG. 2, andwhere d is greater than or equal to c+15.

Preferred epitopes include those comprising a sequence shown in FIGS.1A-B as residues: Asp-30 to Glu-57.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofTR21 and TR22 polypeptides or peptides by recombinant techniques.

The invention further provides an isolated TR21 and TR22 polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

The present invention also provides diagnostic assays such asquantitative and diagnostic assays for detecting levels of TR21 and TR22protein. Thus, for instance, a diagnostic assay in accordance with theinvention for detecting over-expression of TR21 and TR22, or solubleform thereof, compared to normal control tissue samples may be used todetect the presence of tumors.

The TR21 gene is expressed in fetal liver and spleen, and to a lesserextent in bone marrow, umbilical vein, and T cells.

The TR22 gene is expressed in a myeloid progenitor cell line.

Therefore, TR21 polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, disordersof the immune system, particularly hematopoiesis. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the hematopoiesis and immunedisorders, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,immune, hematopoietic, developmental, and cancerous and wounded tissues)or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in fetal liver/spleen and bone marrow indicatesthat the protein product of this gene is useful for diagnosis andtreatment of hematopoietic and immune disorders. Moreover, the proteinproduct of this gene is useful for the treatment and diagnosis ofhematopoetic related disorders such as anemia, pancytopenia, leukopenia,thrombocytopenia or leukemia since stromal cells are important in theproduction of cells of hematopoietic lineages. The uses include bonemarrow cell ex vivo culture, bone marrow transplantation, bone marrowreconstitution, radiotherapy or chemotherapy of neoplasia. The geneproduct may also be involved in lymphopoiesis, therefore, it can be usedin immune disorders such as infection, inflammation, allergy,immunodeficiency etc. In addition, this gene product may have commercialutility in the expansion of stem cells and committed progenitors ofvarious blood lineages, and in the differentiation and/or proliferationof various cell types. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

More specifically, Tumor Necrosis Factor (TNF) family ligands are knownto be among the most pleiotropic cytokines, inducing a large number ofcellular responses, including cytotoxicity, anti-viral activity,immunoregulatory activities, and the transcriptional regulation ofseveral genes. Cellular response to TNF-family ligands include not onlynormal physiological responses, but also diseases associated withincreased apoptosis or the inhibition of apoptosis. Apoptosis-programmedcell death is a physiological mechanism involved in the deletion ofperipheral T lymphocytes of the immune system, and its dysregulation canlead to a number of different pathogenic processes. Diseases associatedwith increased cell survival, or the inhibition of apoptosis, includecancers, autoimmune disorders, viral infections, inflammation, graft vs.host disease, acute graft rejection, and chronic graft rejection.Diseases associated with increased apoptosis include AIDS,neurodegenerative disorders, myelodysplastic syndromes, ischemic injury,toxin-induced liver disease, septic shock, cachexia, and anorexia.

Thus, the invention further provides a method for inhibiting apoptosisinduced by a TNF-family ligand, which involves administering to a cellwhich expresses the TR21 and TR22 polypeptide an effective amount of anagonist capable of increasing TR21 and TR22 mediated signaling.Preferably, TR21 and TR22 mediated signaling is increased to treat adisease wherein increased apoptosis is exhibited.

In a further aspect, the present invention is directed to a method forenhancing apoptosis induced by a TNF-family ligand, which involvesadministering to a cell which expresses the TR21 and TR22 polypeptide aneffective amount of an antagonist capable of decreasing TR21 and TR22mediated activity. Preferably, TR21 and TR22 mediated activity isdecreased to treat a disease wherein decreased apoptosis is exhibited.

Whether any candidate “agonist” or “antagonist” of the present inventioncan enhance or inhibit apoptosis can be determined using art-knownTNF-family ligand/receptor cellular response assays, including thosedescribed in more detail below. Thus, in a further aspect, a screeningmethod is provided for determining whether a candidate agonist orantagonist is capable of enhancing or inhibiting a cellular response toa TNF-family ligand. The method involves contacting cells which expressthe TR21 and TR22 polypeptide with a candidate compound and a TNF-familyligand, assaying a cellular response, and comparing the cellularresponse to a standard cellular response, the standard being assayedwhen contact is made with the ligand in absence of the candidatecompound, whereby an increased cellular response over the standardindicates that the candidate compound is an agonist of theligand/receptor signaling pathway and a decreased cellular responsecompared to the standard indicates that the candidate compound is anantagonist of the ligand/receptor signaling pathway. By the invention, acell expressing the TR21 and TR22 polypeptide can be contacted witheither an endogenous or exogenously administered TNF-family ligand.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B show the nucleotide (SEQ ID NO:1) and deduced amino acid (SEQID NO: 2) sequence of the TR21 receptor. Amino acids 1-29 constitute thesignal peptide.

FIG. 2 shows the partial nucleotide (SEQ ID NO:3) and deduced partialamino acid (SEQ ID NO:4) sequence of the TR22 receptor.

FIGS. 3A-E demonstrates a nucleotide sequence alignment showing theregions of similarity between the nucleotide sequences of the TR21receptor cDNA (FIGS. 1A-B), and the TR22 receptor cDNA (FIG. 2). Regionsof identity are shaded in black. The alignment was performed using theClustal method with Weighted residue weight table.

FIG. 4 shows an amino acid sequence alignment showing the regions ofsimilarity between the amino acid sequences of the TR21 receptor protein(FIGS. 1A-B), and the TR22 receptor protein (FIG. 2). Regions ofidentity are shaded in black. The alignment was performed using theClustal method with PAM250 residue weight table.

FIG. 5 shows an analysis of the TR21 amino acid sequence, determinedusing the various modules and algorithms of the DNA*STAR program set ondefault parameters. Alpha, beta, turn and coil regions; hydrophilicityand hydrophobicity; amphipathic regions; flexible regions; antigenicindex and surface probability are shown. The specific residues definingeach region/fragment depicted in this figure are set forth in tabularform in Table 1, below.

FIG. 6 shows an analysis of the TR22 amino acid sequence, determinedusing the various modules and algorithms of the DNA*STAR program set ondefault parameters. Alpha, beta, turn and coil regions; hydrophilicityand hydrophobicity; amphipathic regions; flexible regions; antigenicindex and surface probability are shown. The specific residues definingeach region/fragment depicted in in this figure are set forth in tabularform in Table 2, below.

FIG. 7 demonstrates that transient expression of transfected TR21 DNA in293T cells, in the presence of a NF-kB-SEAP reporter construct,activated the NF-kB transcription complex in a dose dependent manner,indicating that TR21 plays a role as a cellular proliferative factor.TR11 was added as a positive control.

DETAILED DESCRIPTION OF THE INVENTION

TR21 and TR22 are novel receptor proteins that are highly homologous toeach other, and are members of the tumor necrosis factor (TNF) receptorfamily, with a high degree of homology to TR12 (InternationalPublication No. WO 00/23572). The present invention provides isolatednucleic acid molecules comprising or, alternatively, consisting of apolynucleotide encoding encoding TR21 and TR22 polypeptides having theamino acid sequence shown in FIGS. 1A-B and FIG. 2, respectively. TheTR21 nucleotide sequence shown in FIGS. 1A-B was obtained by cDNA clonenumber HCFMV39 contained in American Type Culture Collection (“ATCC”)Deposit No. 97974, deposited on Apr. 4, 1997. The cDNA of clone numberHCFMV39 is inserted in the pSport 1 plasmid (Life Technologies,Rockville, Md.). The TR22 nucleotide sequence shown in FIG. 2 wasobtained by cDNA clone number HMUCL01 contained in American Type CultureCollection (“ATCC”) Deposit No. PTA-2259, deposited on Jul. 25, 2000.The cDNA of clone number HUMCL01 is inserted in the pCMV SPORT 3.0vector (Life Technologies, Gaithersburg, Md.).

As shown in FIG. 4, the amino acid sequence from Y-58 to T-90 of TR21(FIGS. 1A-B) is highly homologous to the amino acid sequence from Y-7 toT-39 of TR22 (FIG. 2). These segments are also highly homologous to thetransmembrane domain of TR12 (International Publication No. WO00/23572), and thus correspond to highly conserved transmembranedomain-containing segments of TR21 and TR22, resepctively. Thehydrophobicity of the transmembrane regions within these conservedsegments is shown in FIG. 5 and Table 1 for TR21 (see residues fromabout Ile-59 to about Leu 80) and FIG. 6 and Table 2 for TR22 (seeresidues from about Leu-6 to about Val-29).

The intracellular domain of TR21 is from about Leu-81 to through theC-terminus of the protein (Glu-271). Thus Leu-81 through Glu-271 ofFIGS. 1A-B is a specific intracellular fragment of TR21 encompassed bythe invention. The intracellular domain for TR22 is from about Leu-30through the C-terminus of the protein. Thus Leu-30 through Val-194 ofFIG. 2 is a specific intracellular fragment of TR22 encompassed by theinvention. As shown in FIG. 4, the TR21 and TR22 intracellular domainsalso contain homologous regions, including, for example, Arg-215 toThr-231 of TR21 (FIGS. 1A-B) and Arg-146 to Thr-163 of TR22 (FIG. 2).Specific embodiments of the invention contain these conserved regions ofthe TR21 and TR22 polypeptides.

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Using the information provided herein, such as the nucleic acid sequenceset out in FIGS. 1A-B and 2, a nucleic acid molecule of the presentinvention encoding a TR21 or TR22 polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. The TR21 gene is expressed infetal liver and spleen, and to a lesser extent in bone marrow, umbilicalvein, and T cells.

The determined nucleotide sequence of the TR21 cDNA of FIGS. 1A-Bcontains an open reading frame encoding a protein of about 271 aminoacid residues, with a predicted leader sequence of about 29 amino acid.The amino acid sequence of the predicted mature TR21 receptor is shownin FIGS. 1A-B from amino acid residue about 30 to residue about 271. Ofknown members of the TNF receptor family, the TR21 polypeptide of theinvention shares the greatest degree of homology with human TR12 (WO00/23572), including significant sequence homology over multiplecysteine rich domains.

As indicated, the present invention also provides the mature form(s) ofthe TR21 and TR22 receptor of the present invention. According to thesignal hypothesis, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Most mammalian cells and even insect cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species on the protein. Further, it haslong been known that the cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.

Therefore, the present invention provides a nucleotide sequence encodingthe mature TR21 protein, having the amino acid sequence encoded by thecDNA clone HCFMV39 contained in ATCC Deposit No. 97974, or contained inATCC Deposit No. 209080, and as shown in FIGS. 1A-B, and the mature TR22polypeptide, having the amino acid sequence encoded by the cDNA cloneHMUCL01 contained in ATCC Deposit No. PTA-2259. By the mature TR21 andTR22 protein having the amino acid sequence encoded by the cDNA clonescontained in the ATCC deposit numbers specified above is meant themature form(s) of the TR21 and TR22 receptor produced by expression in amammalian cell (e.g., COS cells, as described below) of the completeopen reading frame encoded by the human DNA sequence of the clonecontained in the vector in the deposited host. As indicated below, themature TR21 receptor having the amino acid sequence encoded by therespective cDNA clones contained in the specified ATCC deposit numbers,may or may not differ from the predicted amino acid sequences for themature TR21 protein as shown in FIGS. 1A-B (amino acids 30-271),depending on the accuracy of the predicted cleavage site based oncomputer analysis.

Methods for predicting whether a protein has a secretory leader as wellas the cleavage point for that leader sequence are available. Forinstance, the method of McGeoch (Virus Res. 3:271-286 (1985)) and vonHeinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. vonHeinje, supra. However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

In the present case, the predicted amino acid sequence of the completeTR21 polypeptide of the present invention was analyzed by a computerprogram (“PSORT”). See K. Nakai and M. Kanehisa, Genomics 14:897-911(1992). PSORT is an expert system for predicting the cellular locationof a protein based on the amino acid sequence. As part of thiscomputational prediction of localization, the methods of McGeoch and vonHeinje are incorporated. The analysis by the PSORT program predicted thecleavage site between amino acids 29 and 30 in FIGS. 1A-B. Thereafter,the complete amino acid sequences were further analyzed by visualinspection, applying a simple form of the (−1,−3) rule of von Heinje.von Heinje, supra. Thus, the leader sequence for the TR21 protein ispredicted to consist of amino acid residues from about 1 to about 29 inFIGS. 1A-B, while the mature TR21 protein is predicted to consist ofresidues from about 30-271 in FIGS. 1A-B.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors, as well as the variability of cleavage sites forleaders in different known proteins, the predicted TR21 polypeptideencoded by the deposited cDNA comprises about 271 amino acids, but maybe anywhere in the range of 261-281 amino acids; and the predictedleader sequence of this protein is about 29 amino acids, but may beanywhere in the range of about 19 to about 39 amino acids. It willfurther be appreciated that, the domains described herein have beenpredicted by computer analysis, and accordingly, that depending on theanalytical criteria used for identifying various functional domains, theexact “address” of, for example, the extracellular domain, intracellulardomain, partial death domain, cysteine-rich motifs, and transmembranedomain of TR21 and TR22 may differ slightly. In any event, as discussedfurther below, the invention further provides polypeptides havingvarious residues deleted from the N-terminus and/or C-terminus of thecomplete TR21 and TR22, including polypeptides lacking one or more aminoacids from the N-termini of the extracellular domain described herein,which constitute soluble forms of the extracellular domain of the TR21and TR22 polypeptides.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA may be the coding strand, also known as the sensestrand, or it may be the non-coding strand, also referred to as theanti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.However, a nucleic acid molecule contained in a clone that is a memberof a mixed clone library (e.g., a genomic or cDNA library) and that hasnot been isolated from other clones of the library (e.g., in the form ofa homogeneous solution containing the clone without other members of thelibrary) or a chromosome isolated or removed from a cell or a celllysate (e.g., a “chromosome spread”, as in a karyotype), is not“isolated” for the purposes of this invention.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising or, alternatively, consisting of an open readingframe (ORF) shown in FIGS. 1A-B and 2; DNA molecules comprising or,alternatively, consisting of the coding sequence for the mature TR21 orTR22 protein; and DNA molecules comprising or, alternatively, consistingof a sequence substantially different from those described above, butwhich, due to the degeneracy of the genetic code, still encode the TR21or TR22 protein. Of course, the genetic code is well known in the art.Thus, it would be routine for one skilled in the art to generate suchdegenerate variants.

In another aspect, the invention provides isolated nucleic acidmolecules encoding the TR21 polypeptide having an amino acid sequence asencoded by the cDNA clone contained in the plasmid HCFMV39 contained inATCC Deposit No. 97974, or contained in ATCC Deposit No. 209080, and theTR22 polypeptide having an amino acid sequence as encoded by the cDNAclone HMUCL01 contained in ATCC Deposit No. PTA-2259. In a furtherembodiment, nucleic acid molecules are provided that encode the matureTR21 or TR22 polypeptide or the full length TR21 or TR22 polypeptidelacking the N-terminal methionine. The invention further provides anisolated nucleic acid molecule having the nucleotide sequence shown inFIGS. 1A-B (TR21) or in FIG. 2 (TR22), or the nucleotide sequence of theTR21 or TR22 cDNA contained in the deposited clones described above, ora nucleic acid molecule having a sequence complementary to one of theabove sequences. Such isolated molecules, particularly DNA molecules,are useful as probes for gene mapping by in situ hybridization withchromosomes, and for detecting expression of the TR21 or TR22 gene inhuman tissue, for instance, by Northern blot analysis.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the deposited cDNAs orthe nucleotide sequences shown in FIGS. 1A-B and 2 is intended DNAfragments at least about 15 nt, and more preferably at least about 20nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50-1500 nt in length are also useful according to the presentinvention, as are fragments corresponding to most, if not all, of thenucleotide sequences of the deposited cDNAs or as shown in FIGS. 1A-Band 2. By a fragment at least 20 nt in length, for example, is intendedfragments which include 20 or more contiguous bases from the nucleotidesequence of the deposited cDNAs or the nucleotide sequences as shown inFigures FIGS. 1A-B and 2. In this context “about” includes theparticularly recited size, larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini.

Representative examples of TR21 polynucleotide fragments of theinvention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to 50, 51to 108, 109 to 159, 160 to 210, 211 to 261, 262 to 273, 274 to 324, 325to 375, 376 to 426, 427 to 477, 478 to 528, 529 to 579, 580 to 630, 631to 681, 682 to 732, 733 to 744, 745 to 798, 799 to 849, 850 to 900, 901to 951, 952 to 1002, 1003 to 1053, 1054 to 1104, 1105 to 1155, 1156 to1164, 1165 to 1197, 1198 to 1248, 1249 to 1266, and/or 1267 to 1291 ofFIGS. 1A-B, or the complementary strand thereto, or the cDNA containedin the deposited clone. In this context “about” includes theparticularly recited ranges, larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini.Polynucleotides that hybridize to these polynucleotide fragments arealso encompassed by the invention.

Representative examples of TR22 polynucleotide fragments of theinvention include, for example, fragments that comprise, oralternatively, consist of, a sequence from aboutnucleotide 1 to 50,51 to108,109 to 159, 160to210,211 to 261,262 to 273, 274 to 324, 325 to 375,376 to 426, 427 to 477, 478 to 528, and/or 529 to 582 of FIG. 2, or thecomplementary strand thereto, or the cDNA contained in the depositedclone. In this context “about” includes the particularly recited ranges,larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini. Polynucleotides that hybridize to thesepolynucleotide fragments are also encompassed by the invention.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates a TR21 or TR22 functional activity. By apolypeptide demonstrating a TR21 or TR22 “functional activity” is meant,a polypeptide capable of displaying one or more known functionalactivities associated with a full-length (complete) TR21 or TR22protein. Such functional activities include, but are not limited to,biological, antigenicity (ability to bind (or compete with a TR21 orTR22 polypeptide for binding) to an anti-TR21 or TR22 antibody),immunogenicity (ability to generate antibody which binds to a TR21 orTR22 polypeptide), ability to form multimers with TR21 or TR22polypeptides of the invention, and ability to bind to a receptor orligand for a TR21 or TR22 polypeptide.

The functional activity of TR21 and TR22 polypeptides, and fragments,variants derivatives, and analogs thereof, can be assayed by variousmethods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length TR21 or TR22 polypeptide for binding toanti-TR21 or TR22 antibody, various immunoassays known in the art can beused, including but not limited to, competitive and non-competitiveassay systems using techniques such as radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays, immunoradiometricassays, gel diffusion precipitation reactions, immunodiffusion assays,in situ immunoassays (using colloidal gold, enzyme or radioisotopelabels, for example), western blots, precipitation reactions,agglutination assays (e.g., gel agglutination assays, hemagglutinationassays), complement fixation assays, immunofluorescence assays, proteinA assays, and immunoelectrophoresis assays, etc. In one embodiment,antibody binding is detected by detecting a label on the primaryantibody. In another embodiment, the primary antibody is detected bydetecting binding of a secondary antibody or reagent to the primaryantibody. In a further embodiment, the secondary antibody is labeled.Many means are known in the art for detecting binding in an immunoassayand are within the scope of the present invention.

In another embodiment, where a TR21 and TR22 ligand is identified, orthe ability of a polypeptide fragment, variant or derivative of theinvention to multimerize is being evaluated, binding can be assayed,e.g., by means well-known in the art, such as, for example, reducing andnon-reducing gel chromatography, protein affinity chromatography, andaffinity blotting. See generally, Phizicky, E., et al., Microbiol. Rev.59:94-123 (1995). In another embodiment, physiological correlates ofTR21 and TR22 binding to their substrates (signal transduction) can beassayed.

In addition, assays described herein and otherwise known in the art mayroutinely be applied to measure the ability of TR21 and TR22polypeptides and fragments, variants derivatives and analogs thereof toelicit TR21 or TR22 related biological activity. For example, techniquesknown in the art (such as for example assaying for thymidineincorporation), may be applied or routinely modified to assay for theability of the compositions of the invention to inhibit proliferation ofhematopoietic cells. Additionally, assays desribed herein (see e.g.,Example 20 and Example 38) and otherwise known in the art may be appliedor routinely modified to assay for the ability of the compositions ofthe invention to inhibit or stimulate B cell proliferation.

Other methods will be known to the skilled artisan and are within thescope of the invention.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding: a polypeptide comprising or,alternatively, consisting of the TR21 or TR22 receptor extracellulardomain; a polypeptide comprising or, alternatively, consisting of theTR21 or TR22 cysteine rich domain; a polypeptide comprising or,alternatively, consisting of the TR21 or TR22 transmembrane domain; apolypeptide comprising or, alternatively, consisting of the TR21 or TR22intracellular domain; and a polypeptide comprising or, alternatively,consisting of the incomplete TR21 or TR22 death domain. Since thelocation of these domains have been predicted by computer analysis, oneof ordinary skill would appreciate that the amino acid residuesconstituting these domains may vary slightly (e.g., by about 1 to 15amino acid residues) depending on the criteria used to define eachdomain.

Preferred nucleic acid fragments of the invention encode a full-lengthTR21 or TR22 polypeptide lacking the nucleotides encoding the aminoterminal methionine (nucleotides 1-3 in FIGS. 1A-B), as it is known thatthe methionine is cleaved naturally and such sequences may be useful ingenetically engineering TR21 or TR22 expression vectors. Polypeptidesencoded by such polynucleotides are also contemplated by the invention.

Preferred nucleic acid fragments of the present invention furtherinclude nucleic acid molecules encoding epitope-bearing portions of theTR21 or TR22 receptor protein. Preferred epitopes include thosecomprising a sequence shown in FIG. 1A as residues Asp-30 to Glu-57.Accordingly, such nucleic acid fragments of the present inventioninclude nucleic acid molecules encoding the following TR21 polypeptides:a polypeptide comprising or, alternatively, consisting of amino acidresidues from about 30 to about 57 in FIGS. 1A-B. The inventors havedetermined that the above polypeptide fragments are antigenic regions ofthe TR21 and TR22 protein. Methods for determining other suchepitope-bearing portions of the TR21 and TR22 protein are described indetail below.

It is believed the extracellular domains of TR21 and TR22 are importantfor interactions between TR21 and TR22 and their ligands.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of TR21 and TR22. Preferred embodiments of theinvention in this regard include fragments that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheetforming regions (“beta-regions”), turn and turn-forming regions(“turn-regions”), coil and coil-forming regions (“coil-regions”),hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions andhigh antigenic index regions of TR21 and TR22.

The data representing the structural or functional attributes of TR21and TR22 set forth in FIGS. 5 and 6 and/or Table 1 and 2, respectively,as described above, were generated using the various modules andalgorithms of the DNA*STAR program set on default parameters. In apreferred embodiment, the data presented in columns VIII, IX, XIII, andXIV of Tables 1 and 2 can be used to determine regions of TR21 and TR22,respectively which exhibit a high degree of potential for antigenicity.Regions of high antigenicity are determined from the data presented incolumns VIII, IX, XIII, and/or XIV by choosing values which representregions of the polypeptide which are likely to be exposed on the surfaceof the polypeptide in an environment in which antigen recognition mayoccur in the process of initiation of an immune response. Methods ofmaking and using these antigenic, epitope-containing polypeptidefragments are described in detail below in the section entitled“Epitopes”.

Certain preferred regions in these regards are set out in FIGS. 5 and 6,but may, as shown in Tables 1 and 2, respectively, be represented oridentified by using tabular representations of the data presented inFIGS. 5 and 6. The DNA*STAR computer algorithm used to generate FIGS. 5and 6 (set on the original default parameters) was used to present thedata in FIGS. 5 and 6 in a tabular format (See Tables 1 and 2). Thetabular format of the data in FIGS. 5 and 6 may be used to easilydetermine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIGS. 5 and 6 and inTables 1 and 2, respectively, include, but are not limited to, regionsof the aforementioned types identified by analysis of the amino acidsequence set out in FIGS. 1 and 2. As set out in FIGS. 5 and 6 and inTables 1 and 2, such preferred regions include Garnier-Robsonalpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasmanalpha-regions, beta-regions, and turn-regions, Kyte-Doolittlehydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha-and beta-amphipathic regions, Karplus-Schulz flexible regions,Jameson-Wolf regions of high antigenic index and Emini surface-formingregions.

In specific embodiments relating to TR21, polypeptides of the presentinvention comprise, or alternatively consist of, one, two, three, four,five, six, seven, eight, nine, or all nine of the immunogenic epitopesof the TR-21 protein shown in FIGS. 1A-B as residues: Pro-29 to Pro-56,Leu-81 to Arg-87, Ser-109 to Thr-116, Ser-142 to Pro-149, Gly-168 toLys-173, Cys-192 to Arg-199, Lys-204 to Gly-218, Val-233 to Asn-238, andPro-262 to Glu-271. Fragments and/or variants of these polypeptides,such as, for example, fragments and/or variants as described herein, areencompassed by the invention. Polynucleotides encoding thesepolypeptides (including fragments and/or variants) are also encompassedby the invention, as are antibodies that bind these polypeptides.

In specific embodiments relating to TR22, polypeptides of the presentinvention comprise, or alternatively consist of, one, two, three, four,five, six, or all six of the immunogenic epitopes of the TR-22 proteinshown in FIG. 2 as residues: Leu-30 to Thr-62, Ser-102 to Pro-109,Cys-126 to Pro-133, Gln-137 to Glu-151, His-172 to Gly-185, and Gly-188to Val-194. Fragments and/or variants of these polypeptides, such as,for example, fragments and/or variants as described herein, areencompassed by the invention. Polynucleotides encoding thesepolypeptides (including fragments and/or variants) are also encompassedby the invention, as are antibodies that bind these polypeptides. TABLE1 TR21 Res Pos I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . AB . . . . 0.07 −0.17 * . . 0.45 1.13 Ala 2 . A B . . . . −0.36 −0.10 * .. 0.30 0.90 Pro 3 . A B . . . . −0.18 0.16 * . . −0.30 0.58 Arg 4 . A B. . . . −0.13 0.16 * . . −0.30 0.90 Ala 5 A A . . . . . −0.04 −0.03 * .F 0.45 0.89 Leu 6 . . . . . T C −0.03 −0.14 * . F 1.05 0.77 Pro 7 A . .. . T . −0.30 −0.07 * . F 0.85 0.40 Gly 8 A . . . . T . −0.90 0.57 * . F−0.05 0.29 Ser 9 A . . . . T . −1.60 0.76 * . F −0.05 0.29 Ala 10 A A .. . . . −1.60 0.57 . . . −0.60 0.19 Val 11 A A . . . . . −1.38 0.64 . .. −0.60 0.19 Leu 12 A A . . . . . −2.02 0.71 . . . −0.60 0.15 Ala 13 A A. . . . . −2.38 0.97 . . . −0.60 0.11 Ala 14 A A . . . . . −2.93 1.26 .. . −0.60 0.13 Ala 15 A A . . . . . −2.69 1.26 . . . −0.60 0.11 Val 16 AA . . . . . −2.18 1.00 . . . −0.60 0.11 Phe 17 . A B . . . . −1.96 0.93. . . −0.60 0.11 Val 18 . A B . . . . −2.22 0.93 . . . −0.60 0.11 Gly 19. A B . . . . −1.93 1.07 . . . −0.60 0.11 Gly 20 . . B B . . . −1.640.81 . . . −0.60 0.17 Ala 21 . . . B . . C −1.00 0.41 . . . −0.40 0.30Val 22 . . . B . . C −1.11 0.20 . . F 0.05 0.47 Ser 23 . . B B . . .−1.11 0.46 . . F −0.45 0.39 Ser 24 . . B B . . . −1.36 0.67 . . F −0.450.29 Pro 25 . . B B . . . −1.22 0.67 . . F −0.45 0.39 Leu 26 . . B B . .. −0.63 0.46 . . F −0.45 0.45 Val 27 . . B B . . . 0.22 0.07 . . . −0.300.57 Ala 28 . . B B . . . 0.18 0.09 . . . 0.04 0.59 Pro 29 . . B . . T .0.18 0.09 . . F 0.93 0.71 Asp 30 . . . . T T . 0.09 −0.21 * * F 2.421.28 Asn 31 . . . . T T . 1.01 −0.47 . * F 2.76 1.69 Gly 32 . . . . T T. 1.56 −0.97 . . F 3.40 2.14 Ser 33 . . . . . T C 1.33 −0.91 * . F 2.861.85 Ser 34 . . . . . T C 1.51 −0.23 * . F 2.07 0.95 Arg 35 . . B . . T. 1.21 −0.13 . * F 1.68 1.31 Thr 36 . . B . . T . 1.32 −0.17 . * F 1.341.31 Leu 37 . . B . . . . 1.36 −0.56 . * F 1.10 1.91 His 38 . . B . . T. 1.66 −0.46 * * F 1.30 1.41 Ser 39 . . B . . T . 1.64 −0.46 * * F 1.601.69 Arg 40 . . B . . T . 1.22 −0.46 * * F 1.90 2.95 Thr 41 . . B . . T. 1.32 −0.66 . * F 2.50 3.13 Glu 42 . . . . T . . 1.83 −0.73 . * F 3.003.61 Thr 43 . . B . . . . 1.66 −0.73 . * F 2.30 2.47 Thr 44 . . . . . .C 1.66 −0.30 . * F 2.18 2.65 Pro 45 . . . . . . C 1.54 −0.40 . * F 2.162.05 Ser 46 . . . . . T C 1.86 0.00 . . F 1.74 2.28 Pro 47 . . . . . T C1.54 −0.49 . . F 2.32 2.64 Ser 48 . . . . T T . 1.51 −0.49 . . F 2.802.47 Asn 49 . . . . T T . 1.82 −0.49 . . F 2.52 1.82 Asp 50 . . . . T T. 1.69 −0.47 . . F 2.24 1.89 Thr 51 . . . . T T . 1.96 −0.47 . . F 2.201.40 Gly 52 . . . . T T . 1.96 −0.36 . * F 2.16 1.18 Asn 53 . . . . . TC 2.26 −0.33 . * F 1.92 1.10 Gly 54 . . . . . . C 2.01 −0.33 * . F 1.961.32 His 55 . . . . . T C 1.12 −0.06 * . F 2.40 2.08 Pro 56 . . . . . TC 0.84 0.20 . * F 1.41 0.91 Glu 57 . . B . . T . 0.94 0.30 . * . 0.820.93 Tyr 58 . . B . . T . 0.36 0.63 * . . 0.43 1.07 Ile 59 . . B B . . .−0.11 0.63 . . . −0.36 0.70 Ala 60 . . B B . . . −0.93 0.89 . . . −0.600.33 Tyr 61 . . B B . . . −0.93 1.53 * . . −0.60 0.16 Ala 62 . . B B . .. −1.79 1.20 * . . −0.60 0.35 Leu 63 . . B B . . . −2.24 1.16 . . .−0.60 0.25 Val 64 . . B B . . . −2.06 1.44 . . . −0.60 0.14 Pro 65 . . BB . . . −2.36 1.47 . . . −0.60 0.12 Val 66 . . B B . . . −2.71 1.66 . .. −0.60 0.10 Phe 67 . . B B . . . −2.47 1.59 . . . −0.60 0.14 Phe 68 . .B B . . . −2.47 1.37 . . . −0.60 0.09 Ile 69 . . B B . . . −2.31 1.63 .. . −0.60 0.10 Met 70 . . B B . . . −2.44 1.77 . . . −0.60 0.10 Gly 71 A. . B . . . −2.44 1.41 . . . −0.60 0.11 Leu 72 A . . B . . . −2.56 1.27. . . −0.60 0.12 Phe 73 A . . B . . . −2.74 1.27 . . . −0.60 0.10 Gly 74A . . B . . . −2.52 1.34 * . . −0.60 0.07 Val 75 A . . B . . . −1.961.49 . . . −0.60 0.05 Leu 76 A . . B . . . −2.42 1.30 . . . −0.60 0.07Ile 77 A . . B . . . −2.42 1.20 * . . −0.60 0.06 Cys 78 A . . B . . .−1.68 1.46 * . . −0.60 0.07 His 79 A . . B . . . −1.29 0.81 * . . −0.600.16 Leu 80 A . . B . . . −0.39 0.13 * . . 0.04 0.45 Leu 81 A . . B . .. 0.08 −0.56 * . . 1.43 1.70 Lys 82 . . . B T . . 0.72 −0.70 * . F 2.321.23 Lys 83 . . . . T T . 1.50 −0.44 * . F 2.76 2.34 Lys 84 . . . . T T. 0.87 −1.13 * . F 3.40 5.57 Gly 85 . . . . T T . 1.37 −1.24 * . F 3.061.49 Tyr 86 . . B . . T . 1.87 −0.76 * * . 2.17 1.08 Arg 87 . . B B . .. 1.82 −0.27 * * . 0.98 0.78 Cys 88 . . B B . . . 1.19 −0.27 * * . 0.791.36 Thr 89 . A B B . . . 1.14 −0.20 * * F 0.45 0.88 Thr 90 A A . B . .. 1.49 −0.96 * * F 0.75 0.78 Glu 91 A A . B . . . 1.73 −0.56 * * F 0.902.51 Ala 92 A A . . . . . 0.73 −1.13 * * F 0.90 2.90 Glu 93 A A . . . .. 1.40 −0.93 * * F 0.90 1.41 Gln 94 A A . . . . . 1.71 −1.41 * * F 0.901.41 Asp 95 A A . . . . . 2.02 −1.41 . * F 0.90 2.41 Ile 96 A A . . . .. 2.07 −1.91 . * F 0.90 2.41 Glu 97 A A . . . . . 1.80 −1.91 . * F 0.902.79 Glu 98 A A . . . . . 1.80 −1.67 . . F 0.90 1.24 Glu 99 A A . . . .. 1.84 −1.67 * . F 0.90 3.06 Lys 100 A A . . . . . 0.96 −2.36 * . F 0.903.53 Val 101 A A . . . . . 1.84 −1.67 . * F 0.90 1.43 Glu 102 A A . . .. . 1.03 −1.67 . * F 0.90 1.43 Lys 103 A A . . . . . 1.03 −0.99 . * F0.75 0.59 Ile 104 A A . . . . . 1.03 −0.59 * * F 0.90 1.28 Glu 105 A A .. . . . 0.69 −1.23 . * . 0.75 1.23 Leu 106 A A . . . . . 0.69 −0.84 . *. 0.60 0.83 Asn 107 A . . . . T . 0.69 −0.20 . * . 0.70 0.87 Asp 108 A .. . . T . 0.64 −0.49 . * F 0.85 0.81 Ser 109 . . . . . T C 1.53−0.49 * * F 1.54 1.71 Val 110 . . . . . T C 1.23 −0.77 * * F 2.18 1.71Asn 111 . . . . . . C 2.04 −0.79 * * F 2.32 1.37 Glu 112 . . . . T . .1.73 −0.79 * . F 2.86 1.71 Asn 113 . . . . T T . 0.88 −0.69 * . F 3.403.32 Ser 114 . . . . T T . 0.83 −0.69 * * F 3.06 1.53 Asp 115 . . . . TT . 1.69 −0.66 * * F 2.57 0.87 Thr 116 . . . . T T . 0.80 −0.26 * . F1.93 0.94 Val 117 A . . B . . . −0.06 0.03 * . F 0.19 0.49 Gly 118 . . BB . . . −0.09 0.29 * . F −0.15 0.22 Gln 119 . . B B . . . −0.03 0.79 * .. −0.60 0.21 Ile 120 . . B B . . . −0.92 1.06 * . . −0.60 0.44 Val 121 .. B B . . . −1.21 1.10 * . . −0.60 0.31 His 122 . . B B . . . −0.311.29 * . . −0.60 0.18 Tyr 123 . . B B . . . 0.03 0.89 * . . −0.60 0.50Ile 124 . . B B . . . 0.03 0.60 * . . −0.45 1.09 Met 125 . . B B . . .0.33 −0.04 * * . 0.45 1.39 Lys 126 A . . B . . . 1.19 −0.04 * * . 0.300.89 Asn 127 A A . . . . . 0.63 −0.40 * * F 0.60 2.05 Glu 128 A A . . .. . 0.88 −0.59 . * F 0.90 2.10 Ala 129 A A . . . . . 0.91 −1.20 . * F0.90 1.75 Asn 130 A A . . . . . 0.70 −0.56 . * . 0.60 0.81 Ala 131 A A .. . . . 0.70 −0.27 . * . 0.30 0.38 Asp 132 A A . . . . . 0.11 −0.27 * .. 0.30 0.76 Val 133 A A . . . . . −0.49 −0.27 * * . 0.30 0.48 Leu 134 AA . . . . . −0.76 −0.06 . . . 0.30 0.47 Lys 135 A A . . . . . −1.34 0.09. . . −0.30 0.21 Ala 136 A A . . . . . −0.76 0.59 . . . −0.60 0.28 Met137 A A . . . . . −0.76 −0.06 . . . 0.30 0.57 Val 138 A A . . . . .−0.20 −0.34 . . . 0.30 0.46 Ala 139 A . . . . T . −0.20 0.04 . * . 0.100.61 Asp 140 A . . . . T . −0.49 0.23 . . F 0.25 0.51 Asn 141 . . B . .T . 0.10 0.37 . . F 0.40 1.08 Ser 142 . . B . . T . 0.49 −0.27 . . F1.28 1.78 Leu 143 . . . . . . C 1.34 −0.34 * . F 1.56 1.65 Tyr 144 . . .. . . C 1.63 −0.34 * . F 1.84 1.78 Asp 145 . . . . . T C 1.42 −0.36 * .F 2.32 1.78 Pro 146 . . . . T T . 0.57 −0.31 * . F 2.80 3.33 Glu 147 . .B . . T . 0.56 −0.36 . . F 2.12 1.58 Ser 148 . . B . . T . 1.16 −0.63 .. F 2.14 1.36 Pro 149 . . B B . . . 1.10 −0.20 . * F 1.16 1.36 Val 150 .. B B . . . 0.79 −0.24 . * F 0.88 1.05 Thr 151 . . B B . . . 0.79 0.24 .. F 0.00 1.14 Pro 152 . . B . . . . 0.44 0.29 . . F 0.48 1.14 Ser 153 .. . . T . . 0.44 0.29 . . F 1.16 1.51 Thr 154 . . . . . T C 0.44 0.03 .. F 1.44 1.41 Pro 155 . . . . T T . 1.09 −0.03 . . F 2.52 1.41 Gly 156 .. . . T T . 0.54 −0.03 . . F 2.80 1.62 Ser 157 . . . . . T C 0.46 0.23 .. F 1.57 0.83 Pro 158 . . B . . . . 0.54 0.13 . . F 0.89 0.72 Pro 159 .. B . . . . 0.51 0.13 . . F 0.76 1.13 Val 160 . . B . . . . 0.51 0.13 .. F 0.33 0.83 Ser 161 . . B . . T . 0.04 0.17 . . F 0.25 0.83 Pro 162 .. B . . T . 0.04 0.43 . . F −0.05 0.44 Gly 163 . . B . . T . 0.04 0.39 .. F 0.25 0.80 Pro 164 . . B . . T . −0.09 0.17 . . F 0.25 0.93 Leu 165 .. B . . . . 0.42 0.21 . . F 0.05 0.59 Ser 166 . . B . . T . 0.41 0.21 .. F 0.25 0.59 Pro 167 . . B . . T . 0.41 0.27 . . F 0.25 0.55 Gly 168 .. . . T T . 0.41 0.27 . . F 1.08 1.04 Gly 169 . . . . . T C 0.67 0.01 *. F 1.01 0.77 Thr 170 . . . . . T C 1.44 −0.37 * . F 1.89 0.99 Pro 171 .. . . . T C 0.89 −0.30 * . F 2.32 1.36 Gly 172 . . . . T T . 0.43−0.09 * . F 2.80 1.02 Lys 173 . . B . . T . 0.43 0.06 * . F 1.37 0.38His 174 . . B . . . . 0.74 0.00 . . . 0.74 0.24 Val 175 . . B . . . .1.02 0.07 . . . 0.46 0.33 Cys 176 . . B . . . . 0.42 0.14 . . . 0.180.23 Gly 177 . . B . . . . 0.73 0.83 . . . −0.40 0.14 His 178 . . B . .. . 0.38 0.83 . . . −0.40 0.25 His 179 . . B B . . . −0.44 0.67 * . .−0.60 0.68 Leu 180 . . B B . . . 0.07 0.74 . . . −0.60 0.51 His 181 . .B B . . . 0.39 0.74 . . . −0.60 0.37 Thr 182 . . B B . . . −0.12 0.67 *. . −0.60 0.27 Val 183 . . B B . . . −0.94 0.81 * . . −0.60 0.24 Gly 184. . B B . . . −0.91 0.77 * . . −0.60 0.13 Gly 185 . . B B . . . 0.010.27 * . . −0.30 0.16 Val 186 . . B B . . . 0.04 −0.21 * . . 0.30 0.42Val 187 . . B B . . . −0.50 −0.86 * . . 0.60 0.71 Glu 188 . . B B . . .−0.31 −0.64 * . F 0.75 0.53 Arg 189 . . B . . . . 0.00 −0.50 * * F 0.650.38 Asp 190 A . . . . . . 0.46 −0.64 * * F 0.95 0.70 Val 191 A . . . .. . 0.64 −1.29 * * . 0.80 0.80 Cys 192 A . . . . T . 1.61 −0.71 * * .1.27 0.22 His 193 A . . . . T . 1.58 −0.71 * * . 1.54 0.26 Arg 194 A . .. . T . 1.51 −0.21 * * . 1.51 0.47 Cys 195 A . . . . T . 1.62 −0.86 . *. 2.23 1.75 Arg 196 . . . . T . . 2.19 −1.43 . * . 2.70 2.51 His 197 . .. . T . . 2.82 −1.01 . * . 2.43 1.35 Lys 198 . . . . T . . 2.16 −0.51. * . 2.16 3.42 Arg 199 . . . B T . . 1.16 −0.30 . * . 1.39 1.51 Trp 200. . . B T . . 1.87 0.39 . . . 0.37 0.78 His 201 . . B B . . . 1.54 −0.11. . . 0.30 0.78 Phe 202 . . B B . . . 1.27 0.31 . . . 0.04 0.62 Ile 203. . B B . . . 1.22 0.80 . . . 0.08 0.84 Lys 204 . . . B . . C 1.16 0.29. * F 1.07 1.00 Pro 205 . . . . . T C 1.14 −0.21 * * F 2.56 2.31 Thr 206. . . . T T . 1.29 −0.61 * * F 3.40 4.41 Asn 207 . . . . . T C 1.99−1.30 * * F 2.86 4.32 Lys 208 . . . . . T C 2.58 −1.30 . * F 2.52 4.84Ser 209 . . . . . T C 2.64 −1.34 . * F 2.18 4.49 Arg 210 . . . . T T .2.64 −1.83 . * F 2.38 5.47 Glu 211 . . . . T T . 3.07 −1.80 . . F 2.384.23 Ser 212 . . . . . T C 3.18 −1.80 . . F 2.52 6.18 Arg 213 . . B . .T . 3.13 −2.19 * * F 2.66 6.18 Pro 214 . . . . T T . 3.09 −1.79 * * F3.40 6.18 Arg 215 . . . . T T . 2.98 −1.36 . * F 3.06 4.56 Arg 216 . . .. T T . 2.12 −1.74 * * F 2.72 4.04 Gln 217 . . . B T . . 2.11 −1.10 * *F 1.98 1.94 Gly 218 . . B B . . . 1.14 −1.04 * * F 1.24 1.43 Glu 219 . .B B . . . 0.54 −0.40 . * F 0.45 0.54 Val 220 . . B B . . . 0.13 0.29 * *F −0.15 0.26 Thr 221 . . B B . . . −0.83 0.27 * . . −0.30 0.35 Val 222 .. B B . . . −1.18 0.49 * * . −0.60 0.15 Leu 223 . . B B . . . −0.720.91 * * . −0.60 0.20 Ser 224 . . B . . T . −1.42 0.27 . * . 0.10 0.27Val 225 . . B . . T . −0.46 0.57 . * . −0.20 0.32 Gly 226 . . B . . T .−1.00 −0.07 . * . 0.70 0.75 Arg 227 A . . . . T . −0.46 −0.11 . * . 0.700.42 Phe 228 A . . B . . . 0.40 −0.01 . * . 0.30 0.81 Arg 229 A . . B .. . −0.16 −0.66 . * . 0.75 1.63 Val 230 A . . B . . . 0.70 −0.44 . * .0.30 0.62 Thr 231 . . B B . . . 1.01 −0.44 . * F 0.60 1.24 Lys 232 A . .B . . . 0.94 −0.73 . * F 0.75 0.86 Val 233 A . . . . . . 1.34 −0.73 * *F 1.10 2.31 Glu 234 A . . . . . . 1.23 −0.99 * * F 1.10 2.15 His 235 A .. . . T . 2.09 −1.07 * * F 1.30 1.73 Lys 236 A . . . . T . 2.44 −0.67. * F 1.30 4.03 Ser 237 A . . . . T . 2.40 −1.31 * * F 1.30 4.66 Asn 238A . . . . T . 3.37 −1.31 * * F 1.30 5.93 Gln 239 A A . . . . . 3.48−1.81 * * F 0.90 5.80 Lys 240 A A . . . . . 3.21 −1.81 * * F 0.90 8.48Glu 241 A A . . . . . 2.36 −1.81 * * F 0.90 7.07 Arg 242 A A . . . . .2.06 −1.53 * . F 0.90 3.37 Arg 243 . A B . . . . 1.76 −1.31 * . F 0.901.67 Ser 244 . A B . . . . 0.90 −0.93 * . F 0.90 1.29 Leu 245 . A B . .. . 0.56 −0.29 * * . 0.30 0.49 Met 246 . A B . . . . 0.21 0.10 * . .−0.30 0.33 Ser 247 . . B . . T . −0.49 0.53 * . . −0.20 0.25 Val 248 . .B . . T . −0.60 0.64 * * . −0.20 0.30 Ser 249 . . . . . T C −0.61 −0.04. . F 1.05 0.53 Gly 250 . . B . . T . −0.66 −0.17 . . F 1.11 0.57 Ala251 A . . . . . . −0.06 0.09 . * F 0.57 0.57 Glu 252 A . B . . . . −0.10−0.16 . * F 1.43 0.68 Thr 253 . . B . . T . 0.76 −0.11 * * F 1.89 0.68Val 254 . . B . . T . 0.20 −0.54 * * F 2.60 1.17 Asn 255 . . B . . T .0.33 −0.40 . * F 1.89 0.50 Gly 256 . . B . . T . 0.33 0.03 . * F 1.030.54 Glu 257 . . B . . . . 0.02 0.04 . * F 0.57 0.73 Val 258 . . . . . .C 0.12 −0.11 . * F 1.11 0.66 Pro 259 A . . . . . . 0.12 −0.09 . * F 0.801.03 Ala 260 A . . . . . . 0.17 0.13 * * F 0.05 0.44 Thr 261 . . B . . .. 0.62 0.13 * . F 0.20 1.19 Pro 262 . . B . . . . 0.62 −0.51 * * F 1.101.50 Val 263 . . B . . . . 1.59 −0.94 . . F 1.44 2.58 Lys 264 A . . . .. . 1.50 −1.44 * . F 1.78 3.50 Arg 265 . . B . . . . 1.74 −1.54 . . F2.12 3.03 Glu 266 . . B . . . . 1.74 −1.54 * . F 2.46 4.04 Arg 267 . . .. T T . 1.96 −1.70 * . F 3.40 2.91 Ser 268 . . . . . T C 2.42 −1.70 * .F 2.86 2.58 Gly 269 . . . . . T C 1.99 −1.27 * * . 2.37 1.90 Thr 270 . .. . . T C 1.49 −0.84 . * . 2.03 1.24 Glu 271 . . . . . . C 1.10 −0.41 *. . 1.19 1.18

TABLE 2 TR22 Res Pos I II III IV V VI VII VIII IX X XI XII XIII XIV Arg1 . . . . T . . 1.17 −0.80 . * . 2.59 2.05 Thr 2 . . . . T T . 0.74−0.80 . * . 3.10 1.59 Arg 3 . . . . T T . 0.89 −0.54 . * . 2.79 1.03 Gly4 . . . . T T . 0.68 −0.21 . * . 2.03 0.82 Gly 5 . . . . T T . 0.26 0.40. * F 0.97 0.56 Leu 6 . . . B . . C −0.56 0.60 . * . −0.09 0.24 Tyr 7 .. B B . . . −1.06 1.39 * * . −0.60 0.21 Met 8 . . B B . . . −1.981.64 * * . −0.60 0.17 Leu 9 . . B B . . . −2.49 1.90 . . . −0.60 0.17Phe 10 A . . B . . . −2.96 1.86 . . . −0.60 0.08 Leu 11 A . . B . . .−3.00 1.79 . . . −0.60 0.07 Leu 12 A . . B . . . −3.46 1.81 . . . −0.600.06 Val 13 A . . B . . . −3.56 1.91 . . . −0.60 0.06 Leu 14 A . . B . .. −3.56 1.91 . . . −0.60 0.06 Val 15 A . . B . . . −3.46 1.91 . . .−0.60 0.06 Phe 16 A . . B . . . −2.99 1.84 . . . −0.60 0.09 Phe 17 A . .B . . . −2.99 1.63 . . . −0.60 0.10 Leu 18 A . . B . . . −2.99 1.63 . .. −0.60 0.11 Met 19 A . . B . . . −2.52 1.63 . . . −0.60 0.10 Gly 20 A .. B . . . −2.37 1.27 . . . −0.60 0.11 Leu 21 A . . B . . . −2.27 1.27 .. . −0.60 0.12 Val 22 A . . B . . . −2.46 1.20 . . . −0.60 0.12 Gly 23 A. . B . . . −2.31 1.27 . . . −0.60 0.08 Phe 24 A . . B . . . −1.74 1.41. . . −0.60 0.05 Met 25 A . . B . . . −2.26 1.23 . . . −0.60 0.10 Ile 26A . . B . . . −2.26 1.23 * . . −0.60 0.07 Cys 27 A . . B . . . −1.361.49 * . . −0.60 0.07 His 28 A . . B . . . −0.97 0.70 * . . −0.60 0.14Val 29 A . . B . . . −0.22 0.09 * . . 0.04 0.41 Leu 30 A . . B . . .0.03 −0.60 * . . 1.43 1.53 Lys 31 A . . B . . . 0.68 −0.74 * . F 1.921.11 Lys 32 . . . . T T . 1.46 −0.49 * . F 2.76 2.34 Lys 33 . . . . T T. 0.82 −1.13 * * F 3.40 5.57 Gly 34 . . . . T T . 1.79 −1.24 * * F 3.061.49 Tyr 35 . . . . T T . 2.29 −1.24 * * . 2.57 1.46 Arg 36 . . B B . .. 1.94 −0.76 . . . 1.77 1.05 Cys 37 . . B B . . . 2.01 −0.37 . * . 1.471.43 Arg 38 . . B B . . . 1.62 −0.80 . . F 1.92 1.79 Thr 39 . . . B T .. 1.67 −1.13 . * F 2.51 0.90 Ser 40 . . . . T T . 1.91 −0.74 . * F 3.402.26 Arg 41 . . . . T T . 1.59 −1.31 . * F 3.06 1.99 Gly 42 . . . . T T. 2.26 −0.89 * . F 3.06 2.14 Ser 43 . . . . . T C 2.14 −1.37 . . F 2.862.66 Glu 44 . . . . . T C 1.87 −1.76 . * F 2.86 2.27 Pro 45 . . . . . TC 2.17 −1.26 . * F 2.86 2.32 Asp 46 . . . . T T . 1.24 −1.29 * * F 3.403.00 Asp 47 . . . . T T . 1.59 −0.99 . * F 3.06 1.43 Ala 48 A A . . . .. 1.68 −0.59 . * F 1.92 1.60 Gln 49 A A . . . . . 1.47 −0.59 . . . 1.431.48 Leu 50 . A . . . . C 1.68 −0.16 . * . 1.33 1.37 Gln 51 . A . . . .C 1.68 −0.16 . * F 1.48 2.35 Pro 52 . . . . . T C 1.68 −0.66 . * F 2.522.26 Pro 53 . . . . . T C 2.27 −1.06 . * F 2.86 4.58 Glu 54 . . . . T T. 1.67 −1.74 . * F 3.40 4.42 Asp 55 A . . . . T . 2.48 −1.53 . . F 2.662.83 Asp 56 A . . . . . . 2.48 −1.56 . . F 2.12 2.94 Asp 57 A . . . . .. 2.69 −1.99 . . F 1.78 2.94 Met 58 A . . . . . . 2.59 −1.99 . . F 1.442.94 Asn 59 A . . . . T . 1.73 −1.50 . . F 1.30 2.54 Glu 60 A . . . . T. 1.73 −0.86 * . F 1.30 1.13 Asp 61 A . . . . T . 1.84 −0.86 * * F 1.301.98 Thr 62 A . . . . T . 0.96 −1.47 * * F 1.30 2.41 Val 63 A . . B . .. 0.70 −1.19 * . F 0.75 0.98 Glu 64 A . . B . . . 0.81 −0.54 * . F 0.750.43 Arg 65 A . . B . . . 0.14 −0.54 * . . 0.60 0.59 Ile 66 A . . B . .. −0.74 −0.46 * . . 0.30 0.42 Val 67 A . . B . . . −1.32 −0.41 * . .0.30 0.17 Arg 68 A . . B . . . −0.47 0.27 * . . −0.30 0.06 Cys 69 . . BB . . . −0.47 0.67 * . . −0.60 0.15 Ile 70 . . B B . . . −0.58 0.39 * *. −0.30 0.33 Ile 71 A . . B . . . −0.28 −0.26 * * . 0.30 0.29 Gln 72 A .. B . . . 0.58 0.24 * * . −0.30 0.55 Asn 73 . . . B . . C −0.12 0.07 * *F 0.20 1.26 Glu 74 A . . B . . . 0.54 −0.11 . * F 0.60 1.81 Ala 75 A A .. . . . 0.84 −0.80 . * F 0.90 1.81 Asn 76 A A . . . . . 0.92 −0.70 . * .0.75 1.14 Ala 77 A A . . . . . 0.97 −0.41 . * . 0.30 0.54 Glu 78 A A . .. . . 0.97 −0.41 * . . 0.45 1.07 Ala 79 A A . . . . . 0.37 −0.91 * * .0.75 1.16 Leu 80 A A . . . . . 0.14 −0.70 * . . 0.75 1.13 Lys 81 A A . .. . . −0.20 −0.51 * . . 0.60 0.54 Glu 82 A A . . . . . 0.39 −0.09 * . .0.30 0.53 Met 83 A A . . . . . 0.09 −0.59 * . . 0.75 1.07 Leu 84 A A . .. . . 0.68 −0.89 * . . 0.60 0.72 Gly 85 A A . . . . . 1.14 −0.89 . * F0.75 0.72 Asp 86 A . . . . T . 1.10 −0.46 . * F 0.85 0.72 Ser 87 . . . .. T C 0.76 −1.07 . * F 1.50 1.51 Glu 88 A . . . . T . 1.04 −1.33 . * F1.30 1.51 Gly 89 A . . . . T . 1.00 −1.27 . * F 1.30 1.30 Glu 90 A . . B. . . 1.34 −0.63 . * F 0.75 0.72 Gly 91 A . . B . . . 0.53 −0.61 . * F0.75 0.72 Thr 92 A . . B . . . 0.53 0.07 . * F −0.15 0.60 Val 93 A . . B. . . 0.23 0.03 . * F −0.15 0.47 Gln 94 A . . B . . . −0.28 0.41 . * .−0.60 0.63 Leu 95 . . B B . . . −0.28 0.63 . * . −0.60 0.32 Ser 96 . . BB . . . −0.52 0.14 . * . −0.30 0.73 Ser 97 . . . B . . C −0.52 0.00 . .F 0.05 0.43 Val 98 . . . B . . C 0.03 0.09 . . F 0.05 0.74 Asp 99 . . .B T . . −0.27 −0.21 . * F 0.85 0.74 Ala 100 . . . . . T C −0.27 −0.21. * F 1.05 0.74 Thr 101 . . . . . T C 0.03 0.09 * * F 0.70 0.83 Ser 102. . . . . T C 0.33 −0.16 * * F 1.55 0.86 Ser 103 . . . . . T C 0.84−0.16 * * F 1.95 1.42 Leu 104 . . . . . T C 0.26 −0.23 * . F 2.05 0.97Gln 105 . . . . T T . 0.63 −0.21 * . F 2.50 0.73 Asp 106 . . . . T T .0.64 −0.17 * . F 2.25 0.85 Gly 107 . . . . . T C 0.91 −0.17 * . F 1.951.37 Ala 108 . . . . . T C 1.18 −0.36 * . F 1.70 1.08 Pro 109 . . . . .T C 1.96 −0.26 * . F 1.30 0.88 Ser 110 . . . . . T C 1.64 0.24 * . F0.60 1.21 His 111 A . . . . T . 0.79 0.30 * . . 0.25 1.73 His 112 A . .B . . . 1.10 0.44 . . . −0.60 0.83 His 113 A . . B . . . 0.88 0.51 . . .−0.60 0.84 Thr 114 A . . B . . . 0.74 0.81 . . . −0.60 0.51 Val 115 A .. B . . . 0.74 0.74 . . . −0.60 0.37 His 116 . . . B T . . 0.19 0.63 . .. −0.20 0.37 Leu 117 . . . B T . . −0.37 0.63 . . . −0.20 0.26 Gly 118 .. . B T . . −0.54 0.64 . . . −0.20 0.35 Ser 119 . . . . T . . −0.90 0.43. . . 0.00 0.40 Ala 120 A . . . . . . −0.86 0.50 . . . −0.40 0.26 Ala121 A . . . . . . −0.86 0.50 . . . −0.40 0.21 Pro 122 A . . . . . .−0.71 0.57 . . . −0.40 0.22 Cys 123 A . . . . . . −0.67 0.76 . . . −0.400.12 Leu 124 A . . . . . . −0.26 0.64 . . . −0.06 0.15 His 125 A . . . .. . 0.03 0.14 . * . 0.58 0.19 Cys 126 . . . . T T . 0.67 0.10 . . . 1.520.49 Ser 127 . . . . T T . 0.99 −0.47 * . F 2.76 1.18 Arg 128 . . . . TT . 1.44 −1.16 * . F 3.40 1.69 Ser 129 . . . . T T . 2.04 −1.23 * . F3.06 4.88 Lys 130 . . . . T . . 1.27 −1.37 * . F 2.52 5.64 Arg 131 . . .. . . C 1.08 −1.07 * . F 1.98 2.37 Pro 132 . . . . . . C 1.49 −0.43 . .F 1.34 1.31 Pro 133 . . . . T . . 1.38 −0.81 . . F 1.50 1.29 Leu 134 . .B . . . . 1.33 −0.41 * . F 0.80 1.14 Val 135 . . B . . . . 1.40 0.01 * .F 0.05 0.73 Arg 136 . . B . . T . 0.99 −0.41 . * F 0.85 0.92 Gln 137 A .. . . T . 1.24 −0.46 * . F 1.34 1.50 Gly 138 . . . . . T C 1.46 −1.14 *. F 2.18 4.04 Arg 139 . . . . . T C 1.92 −1.79 * . F 2.52 3.57 Ser 140 .. . . . T C 2.82 −1.36 * . F 2.86 2.04 Lys 141 . . . . T T . 2.41−1.76 * * F 3.40 4.12 Glu 142 . . . . T T . 2.52 −1.80 . * F 3.06 2.82Gly 143 . . . . T T . 2.66 −1.80 . * F 2.72 4.12 Lys 144 . . . . T . .2.66 −1.76 . * F 2.52 3.19 Ser 145 . . . . . . C 2.64 −1.76 * * F 2.323.61 Arg 146 . . . . . T C 2.26 −1.27 * * F 2.52 5.26 Pro 147 . . . . .T C 2.26 −1.27 . * F 2.86 2.60 Arg 148 . . . . T T . 2.29 −1.27 * * F3.40 3.36 Thr 149 . . . . . T C 1.93 −1.17 * * F 2.86 2.48 Gly 150 . . .B . . C 1.38 −0.69 . * F 2.12 2.31 Glu 151 . . . B . . C 0.57 −0.47 . *F 1.33 0.88 Thr 152 . . . B . . C 0.48 0.31 * . F 0.39 0.53 Thr 153 . .B B . . . −0.49 0.21 * . F −0.15 0.71 Val 154 . . B B . . . −0.52 0.43 *. . −0.60 0.31 Phe 155 . . B B . . . −0.07 0.86 * * . −0.60 0.21 Ser 156. . . . . T C −0.77 0.37 . * . 0.30 0.28 Val 157 . . . . . T C −0.340.67 . * . 0.00 0.33 Gly 158 . . . . . T C −0.89 0.03 . * . 0.30 0.75Arg 159 A . . . . T . −0.34 −0.11 . * . 0.70 0.42 Phe 160 A . . B . . .0.32 −0.01 . * . 0.30 0.81 Arg 161 A . . B . . . −0.27 −0.16 . * . 0.451.11 Val 162 A . . B . . . 0.59 0.10 * * . −0.30 0.40 Thr 163 A . . B .. . 0.98 0.10 . * . −0.30 0.79 His 164 A . . B . . . 0.98 −0.69 . * .0.60 0.81 Ile 165 A . . B . . . 1.43 −0.69 * * . 0.75 2.14 Glu 166 A . .B . . . 0.98 −0.57 * * . 0.75 2.32 Lys 167 A . . . . T . 1.02 −0.63 * *F 1.30 1.69 Arg 168 A . . . . T . 1.30 −0.44 . * F 1.00 1.99 Tyr 169 A .. . . T . 1.33 −0.63 . * . 1.15 1.56 Gly 170 A . . . . T . 2.19−0.63 * * . 1.15 1.35 Leu 171 A . . . . . . 2.30 −0.13 * . . 0.84 0.94His 172 A . . . . . . 2.26 −0.13 * . . 1.33 1.18 Glu 173 A . . . . . C1.80 −0.89 * . . 2.17 1.98 His 174 . . . . T T . 1.74 −0.89 * . . 2.912.38 Arg 175 . . . . T T . 1.88 −1.19 * . F 3.40 2.34 Asp 176 . . . . TT . 2.38 −1.26 . . F 3.06 2.09 Gly 177 . . . . T T . 2.41 −0.77 * . F3.00 2.22 Ser 178 . . . . . . C 2.52 −1.27 * . F 2.54 1.89 Pro 179 . . .. . . C 2.26 −1.27 * . F 2.48 2.22 Thr 180 . . . . T . . 1.86 −0.89 * .F 2.62 3.01 Asp 181 . . . . T T . 1.51 −0.40 * . F 2.80 2.36 Arg 182 . .. . T T . 1.56 −0.36 * . F 2.52 1.51 Ser 183 . . . . T T . 1.51 −0.40 *. F 2.24 1.40 Trp 184 . . . . T T . 1.38 −0.46 * . F 1.81 0.83 Gly 185 .. . . . T C 1.34 −0.03 * . F 1.33 0.42 Ser 186 . . . . T T . 1.34 0.40 *. F 0.35 0.31 Gly 187 . . . . T T . 1.23 0.41 * . F 0.35 0.51 Gly 188 .. . . T T . 1.32 −0.50 . . F 1.25 0.86 Gly 189 . . . . . . C 1.27 −0.50. . F 1.07 0.99 Gln 190 . . . . . . C 0.76 −0.46 . . F 1.29 0.99 Asp 191. . . . . T C 0.67 −0.24 . . F 1.71 0.75 Pro 192 . . . . . T C 0.62−0.24 . . . 1.78 0.96 Gly 193 . . . . T T . 0.58 −0.24 . . . 2.20 0.71Val 194 . . B . . T . 0.53 −0.21 . . . 1.58 0.54

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, thecomplement of the TR21 and TR22 coding polynucleotide sequence disclosedherein or the TR21 and TR22 cDNA clones deposited with the ATCC asdescribed above. By “stringent hybridization conditions” is intendedovernight incubation at 42° C. in a solution comprising: 50% formamide,5×SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 g/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers as discussed above and in more detailbelow. In this context “about” includes the particularly recited size,larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in FIGS. 1A-B and 2).

Of course, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly(A) tract of the TR21 cDNA shown in FIG.1B), or to a complementary stretch of T (or U) resides, would not beincluded in a polynucleotide of the invention used to hybridize to aportion of a nucleic acid of the invention, since such a polynucleotidewould hybridize to any nucleic acid molecule containing a poly (A)stretch or the complement thereof (e.g., practically any double-strandedcDNA clone).

In specific embodiments, the polynucleotides of the invention are lessthan 100000 kb, 50000 kb, 10000 kb, 1000 kb, 500 kb, 400 kb, 350 kb, 300kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb, 100 kb, 75 kb, 50 kb, 40 kb,30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 7.5 kb, or 5 kb in length.

In further embodiments, polynucleotides of the invention comprise atleast 15, at least 30, at least 50, at least 100, or at least 250, atleast 500, or at least 1000 contiguous nucleotides of TR21 and TR22coding sequence, but consist of less than or equal to 1000 kb, 500 kb,250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15kb, 10 kb, or 5 kb of genomic DNA that flanks the 5′ or 3′ codingnucleotide set forth in FIGS. 1A-B and 2. In further embodiments,polynucleotides of the invention comprise at least 15, at least 30, atleast 50, at least 100, or at least 250, at least 500, or at least 1000contiguous nucleotides of TR21 and TR22 coding sequence, but do notcomprise all or a portion of any TR21 or TR22 intron. In anotherembodiment, the nucleic acid comprising TR21 and TR22 coding sequencedoes not contain coding sequences of a genomic flanking gene (i.e., 5′or 3′ to the TR21 or TR22 gene in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

As indicated, nucleic acid molecules of the present invention whichencode a TR21 and TR22 polypeptide may include, but are not limited tothe coding sequence for the mature polypeptide, by itself; the codingsequence for the mature polypeptide and additional sequences, such asthose encoding a leader or secretory sequence, such as a pre-, or pro-or prepro-protein sequence; the coding sequence of the maturepolypeptide, with or without the aforementioned additional codingsequences, together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing—including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; additionalcoding sequence which codes for additional amino acids, such as thosewhich provide additional functionalities. Thus, for instance, thepolypeptide may be fused to a marker sequence, such as a peptide, whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The “HA” tag is another peptideuseful for purification which corresponds to an epitope derived from theinfluenza hemagglutinin protein, which has been described by Wilson etal., Cell 37:767-778 (1984). As discussed below, other such fusionproteins include the TR21 and TR22 receptor fused to Fc at the N- orC-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs, orderivatives of the TR21 and TR22 receptor. Variants may occur naturally,such as a natural allelic variant. By an “allelic variant” is intendedone of several alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions which may involve one or more nucleotides. Thevariants may be altered in coding or non-coding regions or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions, or additions.Especially preferred among these are silent substitutions, additions,and deletions, which do not alter the properties and activities of theTR21 and TR22 receptor or portions thereof. Also especially preferred inthis regard are conservative substitutions.

Further embodiments of the invention include isolated nucleic acidmolecules comprising or, alternatively, consisting of a polynucleotidehaving a nucleotide sequence at least 80%, 85%, or 90% identical, andmore preferably at least 95%, 96%, 97%, 98%, or 99% identical to: (a) anucleotide sequence encoding the polypeptide having the amino acidsequence in FIGS. 1A-B and 2; (b) a nucleotide sequence encoding thepolypeptide having the amino acid sequence in FIGS. 1A-B and 2, butlacking the amino terminal methionine; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsabout 1 to about 331 in FIGS. 1A-B and 2; (d) a nucleotide sequenceencoding the polypeptide having the amino acid sequence encoded by theTR21 or TR22 deposited cDNA clones described above, (i.e., contained inATCC Deposit Nos. 97974, 209080, and PTA-2259); (e) a nucleotidesequence encoding the mature TR21 and TR22 polypeptide having the aminoacid sequence encoded by the TR21 and TR22 deposited cDNA clonesdescribed above, (i.e., contained in ATCC Deposit Nos. 97974, 209080,and PTA-2259); (f) a nucleotide sequence encoding the TR21 and TR22receptor extracellular domains; (g) a nucleotide sequence encoding theTR21 or TR22 receptor transmembrane domains; (h) a nucleotide sequenceencoding the TR21 or TR22 receptor intracellular domains; (i) anucleotide sequence encoding the TR21 or TR22 receptor extracellular andintracellular domains with all or part of the transmembrane domaindeleteds; and (j) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i)above.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a TR21 orTR22 polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five mismatches per each 100nucleotides of the reference nucleotide sequence encoding the TR21 orTR22 polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mismatches of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. The reference (query) sequence may be the entireTR21 or TR22 encoding nucleotide sequence shown in FIGS. 1A-B or 2, orany TR21 or TR22 polynucleotide fragment (e.g., a polynucleotideencoding the amino acid sequence of any of the TR21 or TR22 N- and/orC-terminal deletions described herein), variant, derivative or analog,as described herein.

As a practical matter, whether any particular nucleic acid molecule isat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in FIGS. 1A-B and 2 or to thenucleotide sequence of the deposited cDNA clone can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482-489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions, a manual correction ismade to the results to take into consideration the fact that the FASTDBprogram does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example, a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatched/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%. In another example, a 90 base subjectsequence is compared with a 100 base query sequence. This time thedeletions are internal deletions so that there are no bases on the 5′ or3′ of the subject sequence which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only bases 5′ and 3′ of the subject sequencewhich are not matched/aligned with the query sequence are manuallycorrected for. No other manual corrections are made for the purposes ofthis embodiment.

The present application is directed to nucleic acid molecules at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acidsequence for example, shown in FIGS. 1A-B or 2, or to the nucleic acidsequence of the deposited cDNA, irrespective of whether they encode apolypeptide having TR21 or TR22 receptor activity. This is because evenwhere a particular nucleic acid molecule does not encode a polypeptidehaving TR21 or TR22 functional activity, one of skill in the art wouldstill know how to use the nucleic acid molecule, for instance, as ahybridization probe or a polymerase chain reaction (PCR) primer. Uses ofthe nucleic acid molecules of the present invention that do not encode apolypeptide having TR2 1 or TR22 receptor activity include, inter alia:(1) isolating the TR21 or TR22 receptor gene or allelic variants thereofin a cDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of the TR21or TR22 receptor gene, as described in Verma et al., Human Chromosomes.A Manual of Basic Techniques, Pergamon Press, New York (1988); and (3)Northern Blot analysis for detecting TR21 or TR22 receptor mRNAexpression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 80%, 85%, 95%, 96%, 97%, 98% or 99% identical to for example, thenucleic acid sequence shown in FIGS. 1A-B or 2, or to the nucleic acidsequence of the deposited cDNA, which do, in fact, encode a polypeptidehaving TR21 or TR22 receptor functional activity. By “a polypeptidehaving TR21 or TR22 functional receptor activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the TR21 or TR22 receptor of the invention (either thefull-length protein or, preferably, the mature protein), as measured ina particular biological assay. For example, TR21 or TR22 functionalreceptor activity can be measured using the cell death assays performedessentially as previously described (A. M. Chinnaiyan et al., Cell 81:505-512 (1995); M. P. Boldin et al., J. Biol. Chem. 270:7795-8(1995); F.C. Kischkel et al., EMBO 14:5579-5588 (1995); A. M. Chinnaiyan et al.,J. Biol. Chem. 271: 4961-4965 (1996)) and as set forth in Example 5,below. In MCF7 cells, plasmids encoding full-length TR21 areco-transfected with the pLantern reporter construct encoding greenfluorescent protein. Nuclei of cells transfected with TR21 or TR22 willexhibit apoptotic morphology as assessed by DAPI staining. Similar toTNFR-1 and Fas/APO-1 (M. Muzio et al., Cell 85:817-827 (1996); M. P.Boldin et al., Cell 85:803-815 (1996); M. Tewari et al., J. Biol. Chem.270:3255-60 (1995)), TR21 or TR22-induced apoptosis is blocked by theinhibitors of ICE-like proteases, CrmA and z-VAD-fmk. In addition,apoptosis induced by TR21 or TR22 is also blocked by dominant negativeversions of FADD (FADD-DN) or FLICE (FLICE-DN/MACHa1 C360S).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to, for example, the nucleic acidsequence of the deposited cDNA or the nucleic acid sequence shown inFIGS. 1A-B or 2 will encode a polypeptide “having TR21 or TR22 receptorthat, for such nucleic acid molecules that are not degenerate variants,a reasonable number will also encode a polypeptide having TR21 or TR22receptor activity. This is because the skilled artisan is fully aware ofamino acid substitutions that are either less likely or not likely tosignificantly effect protein function (e.g., replacing one aliphaticamino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in J.U. Bowie et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), acid substitutions is provided in J. U.Bowie et al., “Deciphering the Message in Protein substitutions.Polynucleotide assays

This invention is also related to the use of TR21 and TR22polynucleotides to detect complementary polynucleotides such as, forexample, as a diagnostic reagent.

Polynucleotide Assays diagnostic tool that can add or define a diagnosisof a disease or susceptibility to a disease which results fromunder-expression over-expression or altered expression of TR21 or TR22or a soluble form thereof, such as, for example, tumors or autoimmunedisease. (0090] Individuals carrying mutations in the TR21 or TR22 genemay be detected at the DNA level by a variety of techniques. Nucleicacids for diagnosis may be obtained from a patient's cells, such as fromblood, urine, saliva, tissue biopsy and autopsy material.

Individuals carrying mutations in the TR21 or TR22 gene may be detectedat by using PCR prior to analysis. (Saiki et al., Nature 324:163-166(1986)). RNA or cDNA may also be used in the same ways. As an example,PCR primers complementary to the nucleic acid encoding TR21 or TR22 canbe used to identify and analyze TR21 or TR22 by using PCR prior toanalysis. (Saiki et al., Nature 324:163-166 (1986)). RNA or cDNA changein size of the amplified product in comparison to the normal genotype.Point mutations can be identified by hybridizing amplified DNA toradiolabeled TR21 or TR22 RNA or alternatively, radiolabeled TR21 orTR22 antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single- stranded templatemolecule generated by a modified PCR. The sequence determination isexample, a sequencing primer is used with double-stranded PCR product ora single-stranded template molecule generated by a modified PCR. Thesequence determination is

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science 230:1242 (1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and SI protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci.USA 85: 4397-4401 (1985)). Southern blotting of genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinate vectors and/or nucleic acids of theinvention and the production of TR21 and TR22 polypeptides or fragmentsthereof by recombinant techniques.

Host cells can be genetically engineered to incorporate nucleic acidmolecules and express polypeptides of the present invention. Thepolynucleotides may be introduced alone or with other polynucleotides.Such other polynucleotides may be introduced independently,co-introduced or introduced joined to the polynucleotides of theinvention.

In accordance with the present invention the vector may be, for example,a plasmid vector, a single or double-stranded phage vector, a single ordouble-stranded RNA or DNA viral vector. Such vectors may be introducedinto cells as polynucleotides, preferably DNA, by well known techniquesfor introducing DNA and RNA into cells. Viral vectors may be replicationcompetent or replication defective. In the latter case viral propagationgenerally will occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a contain sites for transcripion initiation, termination and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the mature transcripts expressed by the constructswill preferably include a translation initiating at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coll, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl andpSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia. Other suitable vectors will be readily apparent to theskilled artisan.

The present invention also relates to host cells containing theabove-described vector constructs described herein, and additionallyencompasses host cells containing nucleotide sequences of the inventionthat are operably associated with one or more heterologous controlregions (e.g., promoter and/or enhancer) using techniques known of inthe art. The host cell can be a higher eukaryotic cell, such as amammalian cell (e.g., a human derived cell), or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. The host strain may be chosen which modulates theexpression of the inserted gene sequences, or modifies and processes thegene product in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thusexpression of the genetically engineered polypeptide may be controlled.Furthermore, different host cells have characteristics and specificmechanisms for the translational and post-translational processing andmodification (e.g., phosphorylation, cleavage) of proteins. Appropriatecell lines can be chosen to ensure the desired modifications andprocessing of the foreign protein expressed. 101051 Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection orother methods. Such methods are described in many standard laboratorymanuals, such as Davis et al., Basic Methods In Molecular Biology(1986).

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., TR21 or TR22 coding sequence), and/or to includegenetic material (e.g., heterologous polynucleotide sequences) that isoperably associated with TR21 or TR22 polynucleotides of the invention,and which activates, alters, and/or amplifies endogenous TR21 or TR22polynucleotides. For example, techniques known in the art may be used tooperably associate heterologous control regions (e.g., promoter and/orenhancer) and endogenous TR21 or TR22 polynucleotide sequences viahomologous recombination (see, e.g., US Patent Number 5,641,670, issuedJune 24, 1997; International Publication Number WO 96/29411, publishedSeptember 26, 1996; International Publication Number WO 94/12650,published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), thedisclosures of each of which are incorporated by reference in theirentireties).

The polypeptide may be expressed in a modified form, such as a fusionprotein (comprising the polypeptide joined via a peptide bond to aheterologous protein sequence (of a different protein)), and may includenot only secretion signals but also additional heterologous functionalregions. Alternatively, such a fusion protein can be made by proteinsynthetic techniques, e.g., by use of a peptide synthesizer. Thus, aregion of additional amino acids, particularly charged amino acids, maybe added to the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. For example, in specific embodiments,polynucleotides encoding TR21 or TR22 polypeptides of the invention maybe fused to the pelB pectate lyase signal sequence to increase theefficiency to expression and purification of such polypeptides inGram-negative bacteria. See, US Patent Nos. 5,576,195 and 5,846,818, thecontents of which are herein incorporated by reference in theirentireties.

A preferred fusion protein comprises a heterologous region fromimmunoglobulin that is useful to solubilize proteins. For example,EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteinscomprising various portions of constant region of immunoglobin moleculestogether with another human protein or part thereof. In many cases, theFc part in a fusion protein is thoroughly advantageous for use intherapy and diagnosis and thus results, for example, in improvedpharmacokinetic properties (EP-A 0232 262). On the other hand, for someuses, it would be desirable to be able to delete the Fc part after thefusion protein has been expressed, detected and purified in theadvantageous manner described. This is the case when the Fc portionproves to be a hindrance to use in therapy and diagnosis, for example,when the fusion protein is to be used as an antigen for immunizations.In drug discovery, for example, human proteins, such as thehIL5-receptor, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., Journal of Molecular Recognition 8:52-58 (1995) andK. Johanson et al., The Journal of Biological Chemistry 270:16:9459-9471(1995).

The TR21 and TR22 polypeptides of the invention can be recovered andpurified from chemical synthesis and recombinant cell cultures bystandard methods which include, but are not limited to, ammonium sulfateor ethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification. Well knowntechniques for refolding protein may be employed to regenerate activeconformation when the polypeptide is denatured during isolation and/orpurification.

TR21 and TR22 receptor polynucleotides and polypeptides may be used inaccordance with the present invention for a variety of applications,particularly those that make use of the chemical and biologicalproperties of TR21 and TR22. Among these are applications in treatmentof tumors, resistance to parasites, bacteria and viruses, to induceproliferation of T-cells, endothelial cells and certain hematopoieticcells, to treat restenosis, graft vs. host disease, to regulateanti-viral responses and to prevent certain autoimmune diseases afterstimulation of TR21 or TR22 by an agonist. Additional applicationsrelate to diagnosis and to treatment of disorders of cells, tissues andorganisms. These aspects of the invention are discussed further below.Transaenics and “knock-outs”

The TR21 and TR22 proteins of the invention can also be expressed intransgenic animals. Animals of any species, including, but not limitedto, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats,sheep, cows and non-human primates, e.g., baboons, monkeys, andchimpanzees may be used to generate transgenic animals. In a specificembodiment, techniques described herein or otherwise known in the art,are used to express polypeptides of the invention in humans, as part ofa gene therapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., nucleic acids of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., US Patent Number 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, Mol Cell. Biol.3:1803-1814 ( 1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety. Further, the contents of each of the documentsrecited in this paragraph is herein incorporated by reference in itsentirety. Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229(1989), which is incorporated by reference herein in its entirety. Seealso, U.S. Patent No. 5,464,764 (Capecchi, et al., Positive-NegativeSelection Methods and Vectors); U.S. Patent No. 5,631,153 (Capecchi, etal., Cells and Non-Human Organisms Containing Predetermined GenomicModifications and Positive-Negative Selection Methods and Vectors forMaking Same); U.S. Patent No. 4,736,866 (Leder, et al., TransgenicNon-Human Animals); and U.S. Patent No. 4,873,191 (Wagner, et al.,Genetic Transformation of Zygotes); each of which is hereby incorporatedby reference in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each ofwhich is herein incorporated by reference in its entirety).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric animals. The transgene may be integrated as a single transgeneor as multiple copies such as in concatamers, e.g., head-to-head tandemsor head-to-tail tandems. The transgene may also be selectivelyintroduced into and activated in a particular cell type by following,for example, the teaching of Lasko et al. (Proc. NatI. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Science 265:103-106 (1994)). The regulatorysequences required for such a cell-type specific inactivation willdepend upon the particular cell type of interest, and will be apparentto those of skill in the art. The contents of each of the documentsrecited in this paragraph is herein incorporated by reference in itsentirety.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of TR21 or TR22 polypeptides,studying conditions and/or disorders associated with aberrant TR21 orTR22 expression, and in screening for compounds effective inameliorating such conditions and/or disorders.

In further embodiments of the invention, cells that are geneticallyengineered to express the proteins of the invention, or alternatively,that are genetically engineered not to express the proteins of theinvention (e.g., knockouts) are administered to a patient in vivo. Suchcells may be obtained from the patient (i.e., animal, including human)or an MHC compatible donor and can include, but are not limited tofibroblasts, bone marrow cells, blood cells (e.g., lymphocytes),adipocytes, muscle cells, endothelial cells, etc. The cells aregenetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally. Alternatively, the cells can be incorporated into amatrix and implanted in the body, e.g., genetically engineeredfibroblasts can be implanted as part of a skin graft; geneticallyengineered endothelial cells can be implanted as part of a lymphatic orvascular graft. (See, for example, Anderson et al. US Patent Number5,399,349; and Mulligan & Wilson, US Patent Number 5,460,959, each ofwhich is incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system. TR21 andTR22 Receptor Polypeptides and Fragments

The TR21 and TR22 proteins (polypeptides) of the invention may be inmonomers or multimers (i.e., dimers, trimers, tetramers, and highermultimers). Accordingly, the present invention relates to monomers andmultimers of the TR21 and TR22 proteins (polypeptides) of the invention,their preparation, and compositions (preferably, pharmaceuticalcompositions) containing them. In specific embodiments, the polypeptidesof the invention are monomers, dimers, trimers or tetramers. Inadditional embodiments, the multimers of the invention are at leastdimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing only TR21or only TR22 proteins of the invention (including TR21 or TR22fragments, variants, and fusion proteins, as described herein). Thesehomomers may contain TR21 or TR22 proteins having identical or differentpolypeptide sequences. In a specific embodiment, a homomer of theinvention is a multimer containing only TR21 proteins or only TR22proteins having an identical polypeptide sequence. In another specificembodiment, a homomer of the invention is a multimer containing TR21 orTR22 proteins having different polypeptide sequences. In specificembodiments, the multimer of the invention is a homodimer (e.g.,containing TR21 or TR22 proteins having identical or differentpolypeptide sequences) or a homotrimer (e.g., containing TR21 or TR22proteins having identical or different polypeptide sequences). Inadditional embodiments, the homomeric multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containingheterologous proteins (i.e., proteins containing only polypeptidesequences that do not correspond to a polypeptide sequences encoded bythe TR21 gene or the TR22 gene) in addition to the TR21 and TR22proteins of the invention. In a specific embodiment, the multimer of theinvention is a heterodimer, a heterotrimer, or a heterotetramer. Inadditional embodiments, the homomeric multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when proteins of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when proteins of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the TR21 proteins of the invention orthe TR22 proteins of the invention. Such covalent associations mayinvolve one or more amino acid residues contained in the polypeptidesequence of the protein (e.g., the polypeptide sequence recited in FIGS.1A- B or 2 or the polypeptide encoded by the respective deposited cDNAclones). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences ofthe proteins which interact in the native (i.e., naturally occurring)polypeptide. In another instance, the covalent associations are theconsequence of chemical or recombinant manipulation. Alternatively, suchcovalent associations may involve one or more amino acid residuescontained in the heterologous polypeptide sequence in a TR21 or TR22fusion protein. In one example, covalent associations are between theheterologous sequence contained in a fusion protein of the invention(see, e.g., US Patent Number 5,478,925). In a specific example, thecovalent associations are between the heterologous sequence contained ina TR21 or TR22-Fc fusion protein of the invention (as described herein).In another specific example, covalent associations of fusion proteins ofthe invention are between heterologous polypeptide sequences fromanother TNF family ligand/receptor member that is capable of formingcovalently associated multimers, such as for example, oseteoprotegerin(see, e.g., International Publication No. WO 98/49305, the contents ofwhich are herein incorporated by reference in its entirety). In anotherembodiment, two or more TR21 or TR22 polypeptides of the invention arejoined through synthetic linkers (e.g., peptide, carbohydrate or solublepolymer linkers). Examples include those peptide linkers described inU.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteinscomprising multiple TR21 or TR22 polypeptides separated by peptidelinkers may be produced using conventional recombinant DNA technology.

Another method for preparing multimer TR21 or TR22 polypeptides of theinvention involves use of TR21 or TR22 polypeptides fused to a leucinezipper or isoleucine polypeptide sequence. Leucine zipper domains andisoleucine zipper domains are polypeptides that promote multimerizationof the proteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimeric TR21 orTR22 proteins are those described in PCT application WO 94/10308, herebyincorporated by reference. Recombinant fusion proteins comprising asoluble TR21 or TR22 polypeptide fused to a peptide that dimerizes ortrimerizes in solution are expressed in suitable host cells, and theresulting soluble multimeric TR21 or TR22 is recovered from the culturesupernatant using techniques known in the art.

Certain members of the TNF family of proteins are believed to exist intrimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al.,Cell 73:431, 1993). Thus, trimeric TR21 or TR22 may offer the advantageof enhanced biological activity. Preferred leucine zipper moieties arethose that preferentially form trimers. One example is a leucine zipperderived from lung surfactant protein D (SPD), as described in Hoppe etal. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser.No. 08/446,922, hereby incorporated by reference. Other peptides derivedfrom naturally occurring trimeric proteins may be employed in preparingtrimeric TR21 or TR22.

In further preferred embodiments, TR21 or TR22 polynucleotides of theinvention are fused to a polynucleotide encoding a “FLAG” polypeptide.Thus, TR21 and TR22-FLAG fusion proteins are encompassed by the presentinvention. The FLAG antigenic polypeptide may be fused to a TR21 or TR22polypeptide of the invention at either or both the amino or the carboxyterminus. In preferred embodiments, a TR21 or TR22-FLAG fusion proteinis expressed from a pFLAG-CMV-5a or a pFLAG-CMV-1 expression vector(available from Sigma, St. Louis, MO, USA). See, Andersson, S., et al.,J BioL Chem. 264:8222-29 (1989); Thomsen, D. R., et al., Proc. Natl.Acad. Sci. USA, 81:659-63 (1984); and Kozak, M., Nature 308:241 (1984)(each of which is hereby incorporated by reference). In furtherpreferred embodiments, a TR21 or TR22-FLAG fusion protein is detectableby anti-FLAG monoclonal antibodies (also available from Sigma).

In another example, proteins of the invention are associated byinteractions between FLAG polypeptide sequence contained in FLAG-TR21 orTR22 fusion proteins of the invention. In a further embodiment,associated proteins of the invention are associated by interactionsbetween heterologous polypeptide sequence contained in FLAG-TR21 or TR22fusion proteins of the invention and anti-FLAG antibody. 101281 Themultimers of the invention may be generated using chemical techniquesknown in the art. For example, proteins desired to be contained in themultimers of the invention may be chemically cross-linked using linkermolecules and linker molecule length optimization techniques known inthe art (see, e.g., US Patent Number 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, multimers ofthe invention may be generated using techniques known in the art to formone or more inter-molecule cross-links between the cysteine residueslocated within the polypeptide sequence of the proteins desired to becontained in the multimer (see, e.g., US Patent Number 5,478,925, whichis herein incorporated by reference in its entirety). Further, proteinsof the invention may be routinely modified by the addition of cysteineor biotin to the C terminus or N-terminus of the polypeptide sequence ofthe protein and techniques known in the art may be applied to generatemultimers containing one or more of these modified proteins (see, e.g.,US Patent Number 5,478,925, which is herein incorporated by reference inits entirety). Additionally, techniques known in the art may be appliedto generate liposomes containing the protein components desired to becontained in the multimer of the invention (see, e.g., US Patent Number5,478,925, which is herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, proteinscontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., US Patent Number 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., US Patent Number 5,478,925,which is herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain and which can beincorporated by membrane reconstitution techniques into liposomes (see,e.g., US Patent Number 5,478,925, which is herein incorporated byreference in its entirety). 10130] The polypeptides of the presentinvention are preferably provided in an isolated form. By “isolatedpolypeptide” is intended a polypeptide removed from its nativeenvironment. Thus, a polypeptide produced and/or contained within arecombinant host cell is considered isolated for purposes of the presentinvention. Also intended as an “isolated polypeptide” are polypeptidesthat have been purified, partially or substantially, from a recombinanthost cell. For example, a recombinantly produced version of the TR21 orTR22 polypeptide can be substantially purified by the one-step methoddescribed in Smith and Johnson, Gene 67:31-40 (1988). 101311Accordingly, in one embodiment, the invention provides an isolated TR21or TR22 polypeptide having the amino acid sequence encoded by thedeposited cDNA, or the amino acid sequence in FIGS. 1A-B or FIG. 2,respectively, or a peptide or polypeptide comprising a portion of theabove polypeptides.

Polypeptide fragments of the present invention include polypeptidescomprising or alternatively, consisting of, an amino acid sequencecontained in Figures IA-B and 2, encoded by the cDNA contained in thedeposited clone, or encoded by nucleic acids which hybridize (e.g.,under stringent hybridization conditions) to the nucleotide sequencecontained in the deposited clone, or shown in FIGS. 1A-B and 2 or thecomplementary strand thereto. Protein fragments may be “free-standing,”or comprised within a larger polypeptide of which the fragment forms apart or region, most preferably as a single continuous region.Representative examples of polypeptide fragments of the invention,include, for example, fragments that comprise or alternatively, consistof from about amino acid residues: I to 50, 51 to 100, 101 to 157, 158to 175, 176 to 226, and/or 227 to 271 of FIGS. 1A-B; and acid residues:1 to 50, 51 to 100, 101 to 157, 158 to 175, and/or 176 to 194 of FIG. 2.Moreover, polypeptide fragments can be at least 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids inlength. Polynucleotides encoding these polypeptides are also encompassedby the invention. In this context “about” includes the particularlyrecited ranges, larger or smaller by several (5, 4, 3, 2, or 1) aminoacids, at either extreme or at both extremes.

In additional embodiments, the polypeptide fragments of the inventioncomprise, or alternatively consist of, one or more TR21 or TR22 domains.Preferred polypeptide fragments of the present invention include amember selected from the group: (a) a polypeptide comprising oralternatively, consisting of, the TR21 or TR22 extracellular domain; (b)a polypeptide comprising or alternatively, consisting of, the TR21 orTR22 transmembrane domain; (c) a polypeptide comprising oralternatively, consisting of, the TR21 or TR22 intracellular domain; (d)a polypeptide comprising, or alternatively, consisting of, one, two,three, four or more, epitope bearing portions of the TR21 or TR22receptor protein; (e) any combination of polypeptides (a)-(d).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

As discussed above, it is believed that one or both of the extracellulardomains of TR21 and TR22 are important for interactions between TR21 andTR22 and their ligands.

Among the especially preferred fragments of the invention are fragmentscharacterized by structural or functional attributes of TR21 and TR22.Such fragments include amino acid residues that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet andbeta-sheet-fonning regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., containing four or morecontiguous amino acids having an antigenic index of greater than orequal to 1.5, as identified using the default parameters of theJameson-Wolf program) of complete (i.e., full-length) TR21 and TR22(FIGS. 1A-B and 2, respectively). Certain preferred regions are thoseset out in FIG. 5 (TR21) and 6 (TR22) and include, but are not limitedto, regions of the aforementioned types identified by analysis of theamino acid sequence depicted in FIGS. 1A-B and 2, such preferred regionsinclude; Garnier-Robson predicted alpha-regions, beta-regions,turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions,beta-regions, and turn-regions; Kyte-Doolittle predicted hydrophilic andHopp-Woods predicted hydrophobic regions; Eisenberg alpha and betaamphipathic regions; Emini surface-forming regions; and Jameson-Wolfhigh antigenic index regions, as predicted using the default parametersof these computer programs. Polynucleotides encoding these polypeptidesare also encompassed by the invention.

As mentioned above, even if deletion of one or more amino acids from theN-terminus of a protein results in modification of loss of one or morebiological functions of the protein, other functional activities (e.g.,biological activities, ability to multimerize, ability to bind TR21 orTR22 ligand) may still be retained. For example, the ability ofshortened TR21 or TR22 muteins to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptides generallywill be retained when less than the majority of the residues of thecomplete or mature polypeptide are removed from the N-terminus. Whethera particular polypeptide lacking N-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that a TR21 or TR22 mutein with a largenumber of deleted N-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six TR21 or TR22 amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the TR21 aminoacid sequence shown in FIGS. 1A-B, up to the arginine residue atposition number 265 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues nl-271 of FIGS. 1A-B, where n is aninteger from 2 to 265 corresponding to the position of the amino acidresidue in FIGS. 1A-B. Particularly, N-terminal deletions of thepolypeptide can be described by the general formula m-271 where m is aninteger from 2 to 265, where m corresponds to the position of the aminoacid residue identified in FIGS. 1A-B. More in particular, as oneskilled in the art can readily derive from this formula, the inventionprovides polynucleotides encoding polypeptides comprising, oralternatively consisting of, an amino acid sequence selected from thegroup: A-2 to E-271; P-3 to E-271; R-4 to E-271; A-5 to E-271; L-6 toE-271; P-7 to E-271; G-8 to E-271; S-9 to E-271; A-b to E-271; V-Il toE-271; L-12 E-271; A-13 to E-271; A-14 to E-271; A-15 to E-271; V-16 toE-271; F-17 to E-271to E-271; G-19 to E-271; G-20 to E-271; A-21 toE-271; V-22 to E-271; S-23 to E-24 to E-271; P-25 to E-271; L-26 toE-271; V-27 to E-271; A-28 to E-271; P-29 D-30 to E-271; N-31 to E-271;G-32 to E-271; S-33 to E-271; S-34 to E-271; R-35 271; T-36 to E-271;L-37 to E-271; H-38 to E-271; S-39 to E-271; R-40 to E-271;E-271; E-42to E-271; T-43 to E-271; T-44 to E-271; P-45 to E-271; S-46 to E-271toE-271; S-48 to E-271; N-49 to E-271; D-50 to E-271; T-51 to E-271; G-52to E-53 to E-271; G-54 to E-271; H-55 to E-271; P-56 to E-271; E-57 toE-271; Y-58 1-59 to E-271; A-60 to E-271; Y-61 to E-271; A-62 to E-271;L-63 to E-271; V-271; P-65 to E-271; V-66 to E-271; F-67 to E-271; F-68to E-271; 1-69 to E-271;E-271; G-71 to E-271; L-72 to E-271; F-73 toE-271; G-74 to E-271; V-75 to E-271to E-271; 1-77 to E-271; C-78 toE-271; H-79 to E-271; L-80 to E-271; L-81 to E-82 to E-271; K-83 toE-271; K-84 to E-271; G-85 to E-271; Y-86 to E-271; R-87 271; C-88 toE-271; T-89 to E-271; T-90 to E-271; E-91 to E-271; A-92 to E-271;E-271;Q-94 to E-271; D-95 to E-271; 1-96 to E-271; E-97 to E-271; E-98 toE-271to E-271; K-100 to E-271; V-101 to E-271; E-102 to E-271; K-103 toE-271; 1-104 to 271; E-105 to E-271; L-106 to E-271; N-107 to E-271;D-108 to E-271; S-109 to E V-llO toE-271; N-Ill to E-271; E-112 toE-271; N-113 to E-271; S-114 toE-271; D-115 to E-271; T-116 to E-271;V-1 17 to E-271; G-118 to E-271; Q-119 to E-271; 1-120 to 271; V-121 toE-271; H-122 to E-271; Y-123 to E-271; 1-124 to E-271; M-125 to E K-126to E-271; N-127 to E-271; E-128 to E-271; A-129 to E-271; N-130 toE-271; A 131 to E-271; D-132 to E-271; V-133 to E-271; L-134 to E-271;K-135 to E-271; A to E-271; M-137 to E-271; V-138 to E-271; A-139 toE-271; D-140 to E-271; N-141 to 271; S-142 to E-271; L-143 to E-271;Y-144 to E-271; D-145 to E-271; P-146 to E E-147 to E-271; S-148 toE-271; P-149 to E-271; V-150 to E-271; T-151 to E-271; P to E-271; S-153to E-271; T-154 to E-271; P-155 to E-271; G-156 to E-271; S-157 to 271;P-158 to E-271; P-159 to E-271; V-160 to E-271; S-161 to E-271; P-162 toE-G-163 to E-271; P-164 to E-271; L-165 to E-271; S-166 to E-271; P-167to E-271; to E-271; G-169 to E-271; T-170 to E-271; P-171 to E-271;G-172 to E-271; K-173 to 271; H-174 to E-271; V-175 to E-271; C-176 toE-271; G-177 to E-271; H-178 to E H-179 to E-271; L-180 to E-271; H-181to E-271; T-182 to E-271; V-183 to E-271; G to E-271; G-185 to E-271;V-186 to E-271; V-187 to E-271; E-188 to E-271; R-189 to 271; D-190 toE-271; V-191 to E-271; C-192 to E-271; H-193 to E-271; R-194 to E C-195to E-271; R-196 to E-271; H-197 to E-271; K-198 to E-271; R-199 toE-271; W 200 to E-271; H-201 to E-271; F-202 to E-271; 1-203 to E-271;K-204 to E-271; P E-271; T-206 to E-271; N-207 to E-271; K-208 to E-271;S-209 to E-271; R-210 to E 271; E-211 to E-271; S-212 to E-271; R-213 toE-271; P-214 to E-271; R-215 to E R-216 to E-271; Q-217 to E-271; G-218to E-271; E-219 to E-271; V-220 to E-271; T to E-271; V-222 to E-271;L-223 to E-271; S-224 to E-271; V-225 to E-271; G-226 to 271; R-227 toE-271; F-228 to E-271; R-229 to E-271; V-230 to E-271; T-231 to E K-232to E-271; V-233 to E-271; E-234 to E-271; H-235 to E-271; K-236 toE-271; S to E-271; N-238 to E-271; Q-239 to E-271; K-240 to E-271; E-241to E-271; R-242 to 271; R-243 to E-271; S-244 to E-271; L-245 to E-271;M-246 to E-271; S-247 to E V-248 to E-271; S-249 to E-271; G-250 toE-271; A-251 to E-271; E-252 to E-271; T to E-271; V-254 to E-271; N-255to E-271; G-256 to E-271; E-257 to E-271; V-258 to 271; P-259 to E-271;A-260 to E-271; T-261 to E-271; P-262 to E-271; V-263 to E K-264 toE-271; R-265 to E-271 and/or E-266 to E-271 of FIGS. 1A-B. Fragmentsand/or variants of these polypeptides, such as, for example, fragmentsand/or variants as described herein, are encompassed by the invention.Polynucleotides encoding these polypeptides (including fragments and/orvariants) are also encompassed by the invention, as are antibodies thatbind these polypeptides.

In addition, Accordingly, the present invention further providespolypeptides having one or more residues deleted from the amino terminusof the TR22 amino acid sequence shown in FIG. 2, up to the glycineresidue at position number 188 and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues nl-194 of FIG. 2, whereni is an integer from 2 to 188 corresponding to the position of theamino acid residue in FIGS. 1A-B.

More in particular, as the skilled person could readily derive from thefoumula above, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, an amino acidsequence selected from the group: T-2 to V-194; R-3 to V-194; G-4 toV-194; G-5 to V-194; L-6 to V-194; Y-7 to V-194; M-8 to L-9 to V-194;F-10 to V-194; L-11 to V-194; L-12 to V-194; V-13 to V-194; L-14 194;V-15 to V-194; F-16 to V-194; F-17 to V-194; L-18 to V-194; M-19 toV-194;to V-194; L-21 to V-194; V-22 to V-194; G-23 to V-194; F-24 toV-194; M-25 to V-1-26 to V-194; C-27 to V-194; H-28 to V-194; V-29 toV-194; L-30 to V-194; K-194; K-32 to V-194; K-33 to V-194; G-34 toV-194; Y-35 to V-194; R-36 to V-194;to V-194; R-38 to V-194; T-39 toV-194; S-40 to V-194; R-41 to V-194; G-42 to V S-43 to V-194; E-44 toV-194; P-45 to V-194; D-46 to V-194; D-47 to V-194; A-48194; Q-49 toV-194; L-50 to V-194; Q-51 to V-194; P-52 to V-194; P-53 to V-194;toV-194; D-55 to V-194; D-56 to V-194; D-57 to V-194; M-58 to V-194; N-59to V E-60 to V-194; D-61 to V-194; T-62 to V-194; V-63 to V-194; E-64 toV-194; R-65 194; 1-66 to V-194; V-67 to V-194; R-68 to V-194; C-69 toV-194; 1-70 to V-194;V-194; Q-72 to V-194; N-73 to V-194; E-74 to V-194;A-75 to V-194; N-76 to V-194 77 to V-194; E-78 to V-194; A-79 to V-194;L-80 to V-194; K-81 to V-194; E-82 194; M-83 to V-194; L-84 to V-194;G-85 to V-194; D-86 to V-194; S-87 to V-194;to V-194; G-89 to V-194;E-90 to V-194; G-91 to V-194; T-92 to V-194; V-93 to V Q-94 to V-194;L-95 to V-194; S-96 to V-194; S-97 to V-194; V-98 to V-194; D-99 194;A-100 to V-194; T-101 to V-194; S-102 to V-194; S-103 to V-194; L-104 toV Q-105 to V-194; D-106 to V-194; G-107 to V-194; A-108 to V-194; P-109to V-194; S 110 to V-194; H-111 to V-194; H-1 12 to V-194; H-1 13 toV-194; T-114 to V-194; V to V-194; H-116 to V-194; L-117 to V-194; G-118to V-194; S-1 19 to V-194; A-120 to 194; A-121 to V-194; P-122 to V-194;C-123 to V-194; L-124 to V-194; H-125 to V C-126 to V-194; S-127 toV-194; R-128 to V-194; S-129 to V-194; K-130 to V-194; R 131 to V-194;P-132 to V-194; P-133 to V-194; L-134 to V-194; V-135 to V-194; R toV-194; Q-137 to V-194; G-138 to V-194; R-139 to V-194; S-140 to V-194;K-141 to V-194; E-142 to V-194; G-143 to V-194; K-144 to V-194; S-145 toV-194; R-146 to V 194; P-147 to V-194; R-148 to V-194; T-149 to V-194;G-150 to V-194; E-151 to V T-152 to V-194; T-153 to V-194; V-154 toV-194; F-155 to V-194; S-156 to V-194; V 157 to V-194; G-158 to V-194;R-159 to V-194; F-160 to V-194; R-161 to V-194; V to V-194; T-163 toV-194; H-164 to V-194; 1-165 to V-194; E-166 to V-194; K-167 to 194;R-168 to V-194; Y-169 to V-194; G-170 to V-194; L-171 to V-194; H-172 toV E-173 to V-194; H-174 to V-194; R-175 to V-194; D-176 to V-194; G-177to V-194; S 178 to V-194; P-179 to V-194; T-180 to V-194; D-181 toV-194; R-182 to V-194; S to V-194; W-184 to V-194; G-185 to V-194; S-186to V-194; G-187 to V-194; G-188 to V-194 and/or G-189 to V-194 of FIG.2. Fragments and/or variants of these polypeptides, such as, forexample, fragments and/or variants as described herein, are encompassedby the invention. Polynucleotides encoding these polypeptides (includingfragments and/or variants) are also encompassed by the invention, as areantibodies that bind these polypeptides. 10140] Also as mentioned above,even if deletion of one or more amino acids from the C-terminus of aprotein results in modification of loss of one or more biologicalfunctions of the protein, other functional activities (e.g., biologicalactivities (e.g., ability to inhibit TRAIL induced cell death in vivo orin vitro, and/or regulate (e.g., inhibit) B cell proliferation, and/orregulate hematopoiesis; e.g., ability to inhibit the Mixed LymphocyteReaction)), ability to multimerize, ability to bind TR21 or TR22 maystill be retained. For example the ability of the shortened TR21 or TR22mutein to induce and/or bind to antibodies which recognize the completeor mature forms of the polypeptide generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the C-terminus. Whether a particular polypeptidelacking C-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thatan TR21 or TR22 mutein with a large number of deleted C-terminal aminoacid residues may retain some biological or immunogenic activities. Infact, peptides composed of as few as six TR21 or TR22 amino acidresidues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the TR21 polypeptide shown in FIGS. 1A-B, up to the leucineresidue at position number 6, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1r-ml of FIGS. 1A-B,where ml is an integer from 6 to 270 corresponding to the position ofthe amino acid residue in FIGS. 1A-B.

Thus, as one skilled in the art can readily derive from this formula,the invention provides polynucleotides encoding polypeptides comprising,or alternatively consisting of, an amino acid sequence selected from thefollowing group of C-terminal deletions: M-1 to T-270; M-1 to G-269; M-1to S-268; M-1 to R-267; M-1 to E-266; M to R-265; M-1 to K-264; M-1 toV-263; M-1 to P-262; M-1 to T-261; M-1 to A-260;to P-259; M-1 to V-258;M-1 to E-257; M-1 to G-256; M-1 to N-255; M-1 to V-254;to T-253; M-1 toE-252; M-1 to A-251; M-1 to G-250; M-1 to S-249; M-1 to V-248;to S-247;M-1 to M-246; M-1 to L-245; M-1 to S-244; M-1 to R-243; M-1 to R-242;toE-241; M-1 to K-240; M-1 to Q-239; M-1 to N-238; M-1 to S-237; M-1 toK-236;to H-235; M-1 to E-234; M-1 to V-233; M-1 to K-232; M-1 to T-231;M-1 to V-230;to R-229; M-1 to F-228; M-1 to R-227; M-1 to G-226; M-1 toV-225; M-1 to S-224;to L-223; M-1 to V-222; M-l to T-221; M-l to V-220;M-1 to E-219; M-1 to G-218; M-l to Q-217; M-1 to R-216; M-1 to R-215;M-1 to P-214; M-1 to R-213; M-1 to S-212;to E-211; M-1 to R-210; M-1 toS-209; M-1 to K-208; M-1 to N-207; M-1 to T-206;to P-205; M-1 to K-204;M-1 to 1-203; M-l to F-202; M-l to H-201; M-1 to W-200; M to R-199; M-1to K-198; M-1 to H-197; M-1 to R-196; M-1 to C-195; M-1 to R-194;toH-193; M-1 to C-192; M-1 to V-191; M-1 to D-190; M-1 to R-189; M-1 toE-188;to V-187; M-1 to V-186; M-1 to G-185; M-1 to G-184; M-1 to V-183;M-1 to T-182;to H-181; M-1 to L-180; M-1 to H-179; M-1 to H-178; M-1 toG-177; M-1 to C-176;to V-175; M-1 to H-174; M-1 to K-173; M-1 to G-172;M-1 to P-171; M-1 to T-170;to G-169; M-1 to G-168; M-1 to P-167; M-1 toS-166; M-1 to L-165; M-1 to P-164;to G-163; M-1 to P-162; M-1 to S-161;M-1 to V-160; M-1 to P-159; M-1 to P-158;to S-157; M-1 to G-156; M-1 toP-155; M-1 to T-154; M-1 to S-153; M-1 to P-152;to T-151; M-1 to V-150;M-1 to P-149; M-1 to S-148; M-1 to E-147; M-1 to P-146;to D-145; M-1 toY-144; M-1 to L-143; M-1 to S-142; M-1 to N-141; M-1 to D-140;to A-139;M-1 to V-138; M-1 to M-137; M-1 to A-136; M-1 to K-135; M-1 to L-134;toV-133; M-1 to D-132; M-1 to A-131; M-1 to N-130; M-1 to A-129; M-1 toE-128;to N-127; M-1 to K-126; M-1 to M-125; M-1 to 1-124; M-1 to Y-123;M-1 to H-to V-121; M-1 to 1-120; M-1 to Q-119; M-1 to G-118; M-1 to V-117; M-1 to T to D-115; M-1 to S-1 14; M-1 to N-1 13; M-1 to E-112; M-1to N-Ill; M-1 to V-i 10; to S-109; M-1 to D-108; M-1 to N-107; M-1 toL-106; M-1 to E-105; M-1 to 1to K-103; M-1 to E-102; M-1 to V-101; M-1to K-100; M-1 to E-99; M-1 to E-98; E-97; M-1 to 1-96; M-1 to D-95; M-1to Q-94; M-1 to E-93; M-1 to A-92; M-1 to T-90; M-1 to T-89; M-1 toC-88; M-1 to R-87; M-1 to Y-86; M-1 to G K-84; M-1 to K-83; M-1 to K-82;M-1 to L-81; M-1 to L-80; M-1 to H-79; M-1M-1 to 1-77; M-1 to L-76; M-1to V-75; M-1 to G-74; M-1 to F-73; M-1 to G-71; M-1 to M-70; M-1 to1-69; M-1 to F-68; M-1 to F-67; M-1 to V-66; M-1 to V-64; M-1 to L-63;M-1 to A-62; M-1 to Y-61; M-1 to A-60; M-1 to 1Y-58; M-1 to E-57; M-1 toP-56; M-1 to H-55; M-1 to G-54; M-1 to N-53; M-1M-1 to T-51; M-1 toD-50; M-1 to N-49; M-1 to S-48; M-1 to P-47; M-1 to S P-45; M-1 to T-44;M-1 to T-43; M-1 to E-42; M-1 to T-41; M-1 to R-40; M-1 M-1 to H-38; M-1to L-37; M-1 to T-36; M-1 to R-35; M-1 to S-34; M-1 to S G-32; M-1 toN-31; M-1 to D-30; M-1 to P-29; M-1 to A-28; M-1 to V-27; M-1M-1 toP-25; M-1 to S-24; M-1 to S-23; M-1 to V-22; M-1 to A-21; M-1 to G G-19;M-1 to V-18; M-1 to F-17; M-1 to V-16; M-1 to A-15; M-1 to A-14; M-1 M-1to L-12; M-1 to V-il; M-1 to A-10; M-1 to S-9; M-1 to G-8 and/or M-1 toP-7 Figures iA-B. Fragments and/or variants of these polypeptides, suchas, for example, fragments and/or variants as described herein, areencompassed by the invention. Polynucleotides encoding thesepolypeptides (including fragments and/or variants) are also encompassedby the invention, as are antibodies that bind these polypeptides.

In addition, the present invention fuirther provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the TR22 polypeptide shown in FIG. 2, up to the leucineresidue at position number 6, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-ml of FIG. 2, where mlis an integer from 6 to 194 corresponding to the position of the aminoacid residue in FIG. 2. Polynucleotides encoding these polypeptides arealso encompassed by the invention.

Thus, as the skilled person could readily derive from the formula above,the invention provides polynucleotides encoding polypeptides comprising,or alternatively consisting of, an amino acid sequence selected from thefollowing group of C-terminal deletions: R-1 to G-193; R-1 to P-192; R-1to D-191; R-l to Q-190; R-1 to G-189; R-1 G-188; R-1 to G-187; R-1 toS-186; R-1 to G-185; R-1 to W-184; R-1 to S-183; 182; R-1 to D-181; R-1to T-180; R-1 to P-179; R-1 to S-178; R-1 to G-177; R 176; R-1 to R-175;R-1 to H-174; R-1 to E-173; R-1 to H-172; R-1 to L-171; R 170; R-1 toY-169; R-1 to R-168; R-1 to K-167; R-1 to E-166; R-1 to 1-165; 164; R-Ito T-163; R-1 to V-162; R-1 to R-161; R-1 to F-160; R-1 to R-159; R-1158; R-1 to V-157; R-1 to S-156; R-1 to F-155; R-1 to V-154; R-1 toT-153; R 152; R-1 to E-151; R-1 to G-150; R-1 to T-149; R-1 to R-148;R-1 to P-147; R 146; R-I to S-145; R-1 to K-144; R-1 to G-143; R-1 toE-142; R-1 to K-141; R-1 140; R-1 to R-139; R-1 to G-138; R-1 to Q-137;R-1 to R-136; R-1 to V-135; R 134; R-1 to P-133; R-1 to P-132; R-1 toR-131; R-1 to K-130; R-1 to S-129; R R-1 to S-127; R-1 to C-126; R-1 toH-125; R-1 to L-124; R-1 to C-123; R-1 to to A-121; R-1 to A-120; R-1 toS-1 19; R-1 to G-118; R-1 to L-117; R-1 to H-1 16 V-1 15; R-1 to T-114;R-1 to H-1 13; R-1 to H-1 12; R-1 to H-Il1; R-1 to S-1 10; 109; R-I toA-108; R-I to G-107; R-I to D-106; R-1 to Q-105; R-I to L-104; R-I to S-103; R-1 to S-102; R-1 to T-101; R-1 to A-100; R-1 to D-99; R-1 to V-98;R-1 to S-96; R-1 to L-95; R-1 to Q-94; R-1 to V-93; R-1 to T-92; R-1 toG-91 R-1 to G-89; R-1 to E-88; R-1 to S-87; R-1 to D-86; R-1 to G-85;R-1 to L-83; R-1 to E-82; R-1 to K-81; R-1 to L-80; R-1 to A-79; R-1 toE-78; R-1 N-76; R-1 to A-75; R-1 to E-74; R-1 to N-73; R-1 to Q-72; R-1to 1-71; C-69; R-1 to R-68; R-1 to V-67; R-1 to 1-66; R-1 to R-65; R-1to E-64; to T-62; R-1 to D-61; R-1 to E-60; R-1 to N-59; R-1 to M-58;R-1 to D-57; R R-1 to D-55; R-1 to E-54; R-1 to P-53; R-1 to P-52; R-1to Q-51; R-1 to L-49; R-I to A-48; R-I to D-47; R-I to D-46; R-I toP-45; R-I to E-44; R-I to S-43; R-l to G-42; R-1 to R-41; R-1 to S-40;R-1 to T-39; R-1 to R-38; R-1 to C-37; R-1 to Y-35; R-1 to G-34; R-1 toK-33; R-1 to K-32; R-1 to K-31; R-1 to L-30; R R-1 to H-28; R-1 to C-27;R-1 to 1-26; R-1 to M-25; R-1 to F-24; R-1 to 22; R-1 to L-21; R-1 toG-20; R-1 to M-19; R-1 to L-18; R-1 to F-17; R-1 V-15; R-1 to L-14; R-1to V-13; R-1 to L-12; R-1 to L-ii; R-1 to F-10; R-1 to M-8 and/or R-I toY-7 of FIG. 2. Fragments and/or variants of these polypeptides, such as,for example, fragments and/or variants as described herein, areencompassed by the invention. Polynucleotides encoding thesepolypeptides (including fragments and/or variants) are also encompassedby the invention, as are antibodies that bind these polypeptides.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein (e.g., ability to inhibitthe Mixed Lymphocyte Reaction), other functional activities (e.g.,biological activities, ability to multimerize, ability to bind ligand,ability to generate antibodies, ability to bind antibodies) may still beretained. For example, the ability of the shortened polypeptide toinduce and/or bind to antibodies which recognize the complete or matureforms of the polypeptide generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the C-terminus. Whether a particular polypeptide lackingC-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a polypeptidewith a large number of deleted C-terminal amino acid residues may retainsome biological or immunogenic activities. In fact, peptides composed ofas few as six amino acid residues may often evoke an immune response.Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the polypeptide shown in FIG. 2, as described by the generalformula i-n, where n is an integer from 6 to 194, where n corresponds tothe position of the amino acid residue identified in FIG. 2.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues nl- ml and/or n²- ml of FIGS.1A-B or FIG. 2, where n, n², and m¹ are integers as described above.Thus, any of the above listed N- or C-terminal deletions can be combinedto produce a N- and C-terminal deleted TR21 or TR22 polypeptide.

The invention is also directed to nucleic acid molecules comprising or,alternatively, consisting of a nucleotide sequence at least 80%, 85%,90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotidesequences encoding the TR21 and TR22 polypeptides described above. Theinvention also encompasses these nucleotide sequences fused to aheterologous nucleotide sequence. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention.

It will be recognized in the art that some amino acid sequences of TR21and TR22 can be varied without significant effect on the structure orfunction of the protein. If such differences in sequence arecontemplated, it should be remembered that there will be critical areason the protein which determine activity. Thus, the invention furtherincludes variations of the TR21 and TR22 receptors, which showsubstantial TR21 or TR22 receptor activity or which include regions ofTR21 or TR22 proteins, such as the protein portions discussed herein.Such mutants include deletions, insertions, inversions, repeats, andtype substitutions. As indicated above, guidance concerning which aminoacid changes are likely to be phenotypically silent can be found in J.U.Bowie et aL, Science 247:1306-1310 (1990).

Thus, the fragment, derivative, or analog of the polypeptide of FIGS.1A-B or FIG. 2, or that encoded by the deposited cDNA, may be (i) one inwhich at least one or more of the amino acid residues are substitutedwith a conserved or non-conserved amino acid residue (preferably aconserved amino acid residue(s), and more preferably at least one butless than ten conserved amino acid residues) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asan IgG Fc fusion region peptide or leader or secretory sequence or asequence which is employed for purification of the mature polypeptide ora proprotein sequence. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the TR21 or TR22 receptor protein. Theprevention of aggregation is highly desirable. Aggregation of proteinsnot only results in a loss of activity but can also be problematic whenpreparing pharmaceutical formulations, because they can be immunogenic.(Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al.,Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic DrugCarrier Systems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266-268(1993), describes certain mutations resulting in selective binding ofTNF-(X to only one of the two known types of TNF receptors. Thus, theTR21 and TR22 receptors of the present invention may include one or moreamino acid substitutions, deletions, or additions, either from naturalmutations or human manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table II). TABLE 3Conservative Amino Acid Substitutions Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine101531 In specific embodiments, the number of substitutions, additionsor deletions in the amino acid sequence of FIGS. 1A-B and 2 and/or anyof the polypeptide fragments described herein (e.g., the extracellulardomain or intracellular domain) is 75, 70, 60, 50, 40, 35, 30, 25, 20,15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 30-20, 20-15, 20-10, 15-10, 10-1,5-10, 1-5, 1-3 or 1-2. (01541 Amino acids in the TR21 and TR22 proteinsof the present invention that are essential for function can beidentified by methods known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,Science 244:1081-1085 (1989)). The latter procedure introduces singlealanine mutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity such as receptorbinding or in vitro proliferative activity. Sites that are critical forligand-receptor binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos etal. Science 255:306-312 (1992)).

To improve or alter the characteristics of TR21 and TR22 polypeptides,protein engineering may be employed. Recombinant DNA technology known tothose skilled in the art can be used to create novel mutant proteins or“muteins including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

Non-naturally occurring variants may be produced using art-knownmutagenesis techniques, which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)),restriction selection mutagenesis (see e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

Thus, the invention also encompasses TR21 and TR22 derivatives andanalogs that have one or more amino acid residues deleted, added, orsubstituted to generate TR21 and TR22 polypeptides that are bettersuited for expression, scale up, etc., in the host cells chosen. Forexample, cysteine residues can be deleted or substituted with anotheramino acid residue in order to eliminate disulfide bridges; N-linkedglycosylation sites can be altered or eliminated to achieve, forexample, expression of a homogeneous product that is more easilyrecovered and purified from yeast hosts which are known tohyperglycosylate N-linked sites. To this end, a variety of amino acidsubstitutions at one or both of the first or third amino acid positionson any one or more of the glycosylation recognitions sequences in theTR21 and TR22 polypeptides of the invention, and/or an amino aciddeletion at the second position of any one or more such recognitionsequences will prevent glycosylation of the TR21 and TR22 at themodified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J5(6):1193-1197). Additionally, one or more of the amino acid residues ofthe polypeptides of the invention (e.g., arginine and lysine residues)may be deleted or substituted with another residue to eliminateundesired processing by proteases such as, for example, furins orkexins.

The polypeptides of the present invention include a polypeptidecomprising, or alternatively, consisting of the polypeptide encoded bythe deposited cDNA including the leader; a polypeptide comprising, oralternatively, consisting of the mature polypeptide encoded by thedeposited cDNA minus the leader (i.e., the mature protein); apolypeptide comprising, or alternatively, consisting of thetransmembrane domain; and a polypeptide comprising, or alternatively,consisting of the intracellular domain; a polypeptide comprising, oralternatively, consisting of the extracellular and intracellular domainswith all or part of the transmembrane domain deleted; as well aspolypeptides which are at least 80% identical, more preferably at least80%, 85%, 90%, or 95% identical, still more preferably at least 96%,97%, 98%, or 99% identical to the polypeptides described above (e.g.,the polypeptide encoded by the deposited cDNA clones, the polypeptide ofFigures lA-B or 2, and also include portions of such polypeptides withat least 30 amino acids and more preferably at least 50 amino acids.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a TR21 or TR22polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the TR21 or TR22 receptor. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for instance,the amino acid sequence shown in FIGS. 1A-B or 2, or to the amino acidsequence encoded by the deposited cDNA clone, can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison, WI53711). When using Bestfit or any other sequence alignment program todetermine whether a particular sequence is, for instance, 95% identicalto a reference sequence according to the present invention, theparameters are set, of course, such that the percentage of identity iscalculated over the full length of the reference amino acid sequence andthat gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=l,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N- or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N- and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence. For example, a 90 aminoacid residue subject sequence is aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C- termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N- or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

In additional embodiments, polynucleotides of the invention comprise, oralternatively consist of, a polynucleotide sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequenceencoding the extracellular domain of TR21 or TR22 disclosed in FIGS.1A-B or 2. In another embodiment, the invention provides an isolatednucleic acid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to the TR21 or TR22 extracellulardomain. The present invention also encompasses the abovepolynucleotide/nucleic acid sequences fused to a heterologouspolynucleotide sequence. Polypeptides encoded by these nucleic acidsand/or polynucleotide sequences are also encompassed by the invention.10163] The present application is also directed to proteins cotainingpolypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identicalto the TR21 or TR22 polypeptide sequence set forth as n -mn, and/or n²-ml herein, above. In preferred embodiments, the application is directedto proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to polypeptides having the amino acid sequenceof the specific TR21 or TR22 N- and C-terminal deletions recited herein.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

In certain preferred embodiments, TR21 or TR22 proteins of the inventioncomprise fusion proteins as described above wherein the TR21 or TR22polypeptides are those described as nl-m , and/or n²- ml herein. Inpreferred embodiments, the application is directed to nucleic acidmolecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical tothe nucleic acid sequences encoding polypeptides having the amino acidsequence of the specific N- and C-terminal deletions recited herein.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells using, for example, the recombinant compositions andmethods described above. Depending upon the host employed in arecombinant production procedure, the polypeptides of the presentinvention may be glycosylated or non-glycosylated. In addition,polypeptides of the invention may also include an initial modifiedmethionine residue, in some cases as a result of host-mediatedprocesses. 10166] In addition, proteins of the invention can bechemically synthesized using techniques known in the art (e.g., seeCreighton, Proteins: Structures and Molecular Principles, W.H. Freeman &Co., N.Y. (1983), and Hunkapiller, et al., Nature 310:105-111 (1984)).For example, a peptide corresponding to a fragment of the TR21 or TR22polypeptides of the invention can be synthesized by use of a peptidesynthesizer. Furthermore, if desired, nonclassical amino acids orchemical amino acid analogs can be introduced as a substitution oraddition into the TR21 or TR22 polypeptide sequence. Non-classical aminoacids include, but are not limited to, to the D-isomers of the commonamino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid,4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids,designer amino acids such as b-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The invention additionally, encompasses TR21 and TR22 polypeptides whichare differentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protectingiblocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited to, specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin; etc. 101681 Additional post-translational modificationsencompassed by the invention include, for example, e.g., N-linked or0-linked carbohydrate chains, processing of N-terminal or C-terminalends), attachment of chemical moieties to the amino acid backbone,chemical modifications of N-linked or 0-linked carbohydrate chains, andaddition or deletion of an N-terminal methionine residue as a result ofprocaryotic host cell expression. The polypeptides may also be modifiedwith a detectable label, such as an enzymatic, fluorescent, isotopic oraffinity label to allow for detection and isolation of the protein.

Also providedby the invention are chemically modified derivatives ofTR21 and TR22 which may provide additional advantages such as increasedsolubility, stability and circulating time of the polypeptide, ordecreased immunogenicity (see U. S. Patent No. 4,179,337). The chemicalmoieties for derivitization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000,70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa. 10171]As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. PatentNo. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference. 10172] Thepolyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN- terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues, glutamic acid residues and theC-terminal amino acid residue. Sulfhydryl groups may also be used as areactive group for attaching the polyethylene glycol molecules.Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to a proteins via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N- terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the protein either directly or by an interveninglinker. Linkerless systems for attaching polyethylene glycol to proteinsare described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992); Francis et al., Intern. J of Hematol. 68:1-18 (1998);U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; andWO 98/32466, the disclosures of each of which are incorporated herein byreference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride (CISO₂CH₂CF₃). Upon reaction ofprotein with tresylated MPEG, polyethylene glycol is directly attachedto amine groups of the protein. Thus, the invention includesprotein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Patent No. 5,612,460,the entire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with 1,1-carbonyldiimidazole,MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, andvarious MPEG-succinate derivatives. A number additional polyethyleneglycol derivatives and reaction chemistries for attaching polyethyleneglycol to proteins are described in WO 98/32466, the entire disclosureof which is incorporated herein by reference. Pegylated protein productsproduced using the reaction chemistries set out herein are includedwithin the scope of the invention.

The number of polyethylene glycol moieties attached to each protein ofthe invention (i.e., the degree of substitution) may also vary. Forexample, the pegylated proteins of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 1013, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

As mentioned the TR21 and TR22 proteins of the invention may be modifiedby either natural processes, such as posttranslational processing, or bychemical modification techniques which are well known in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degrees at several sites in a given TR21 or TR22polypeptide. TR21 and TR22 polypeptides may be branched, for example, asa result of ubiquitination, and they may be cyclic, with or withoutbranching. Cyclic, branched, and branched cyclic TR21 and TR22polypeptides may result from posttranslation natural processes or may bemade by synthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993);POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol182:626-646 (1990); Rattan et al., Ann NYAcad Sci 663:48-62 (1992)).Epitopes

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence FIGS. 1A-B and 2, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in the depositedclones HCFMV39 and HMUCL01 or encoded by a polynucleotide thathybridizes to the complement of the nucleotide coding sequence of FIGS.1A-B or 2, or contained deposited clones HCFMV39 and HMUCL01, understringent hybridization conditions or lower stringency hybridizationconditions as defined supra. The present invention further encompassespolynucleotide sequences encoding an epitope of a polypeptide sequenceof the invention (such as, for example, the sequences disclosed FIGS. 1A-B and 2), polynucleotide sequences of the complementary strand of apolynucleotide sequence encoding an epitope of the invention, andpolynucleotide sequences which hybridize to the complementary strandunder stringent hybridization conditions or lower stringencyhybridization conditions defined supra. (01811 The term “epitopes,” asused herein, refers to portions of a polypeptide having antigenic orimmunogenic activity in an animal, preferably a mammal, and mostpreferably in a human. In a preferred embodiment, the present inventionencompasses a polypeptide comprising an epitope, as well as thepolynucleotide encoding this polypeptide. An “immunogenic epitope,” asused herein, is defined as a portion of a protein that elicits anantibody response in an animal, as determined by any method known in theart, for example, by the methods for generating antibodies describedinfra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, isdefined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Specific antigenic epitope fragments of TR21 and TR22 of the inventionare presented in tabular form in Tables 1 and 2, above.

Fragments that finction as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Patent No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, and, most preferably, between about 15 to about 30amino acids. Preferred polypeptides comprising immunogenic or antigenicepitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenicepitopes are usefuil, for example, to raise antibodies, includingmonoclonal antibodies, that specifically bind the epitope. Antigenicepitopes can be used as the target molecules in immunoassays. (See, forinstance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al.,Science 219:660-666 (1983)). 101851 Similarly, immunogenic epitopes canbe used, for example, to induce antibodies according to methods wellknown in the art. (See, for instance, Sutcliffe et al., supra; Wilson etal., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; andBittle et al., J. Gen. Virol. 66:2347-2354 (1985). The polypeptidescomprising one or more immunogenic epitopes may be presented foreliciting an antibody response together with a carrier protein, such asan albumin, to an animal system (such as, for example, rabbit or mouse),or, if the polypeptide is of sufficient length (at least about 25 aminoacids), the polypeptide may be presented without a carrier. However,immunogenic epitopes comprising as few as 8 to 10 amino acids have beenshown to be sufficient to raise antibodies capable of binding to, at thevery least, linear epitopes in a denatured polypeptide (e.g., in Westernblotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as, for example, rabbits, rats, and miceare immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 micrograms of peptide or carrier protein andFreund's adjuvant or any other adjuvant known for stimulating an immuneresponse. Several booster injections may be needed, for instance, atintervals of about two weeks, to provide a useful titer of anti-peptideantibody that can be detected, for example, by ELISA assay using freepeptide adsorbed to a solid surface. The titer of anti-peptideantibodies in serum from an immunized animal may be increased byselection of anti-peptide antibodies, for instance, by adsorption to thepeptide on a solid support and elution of the selected antibodiesaccording to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope described herein can be fused to other polypeptidesequences. For example, the polypeptides of the present invention may befused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM),or portions thereof (CHI, CH2, CH3, or any combination thereof andportions thereof) resulting in chimeric polypeptides. Such fusionproteins may facilitate purification and may increase half-life in vivo.This has been shown for chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins. See,e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). IgGFusion proteins that have a disulfide-linked dimeric structure due tothe IgG portion desulfide bonds have also been found to be moreefficient in binding and neutralizing other molecules than monomericpolypeptides or fragments thereof alone. See, e.g., Fountoulakis et al.,J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the aboveepitopes can also be recombined with a gene of interest as an epitopetag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detectionand purification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix-binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded ontoNi²+nitriloacetic acid-agarose column and histidine-tagged proteins canbe selectively eluted with imidazole containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265- 76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308- 13 (1998) (each of these patentsand publications are hereby incorporated by reference in its entirety).In one embodiment, alteration of polynucleotides corresponding FIGS.1A-B and 2, and the polypeptides encoded by these polynucleotides, maybe achieved by DNA shuffling. DNA shuffling involves the assembly of twoor more DNA segments by homologous or site specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide coding apolypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules. Polypeptide Assays

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of TR21 and TR22receptor protein, or the soluble form thereof, in cells and tissues,including determination of normal and abnormal levels. Thus, forinstance, a diagnostic assay in accordance with the invention fordetecting over expression of TR21 and TR22, or soluble form thereof,compared to normal control tissue samples may be used to detect thepresence of tumors, for example. Assay techniques that can be used todetermine levels of a protein, such as a TR21 and TR22 protein of thepresent invention, or a soluble form thereof, in a sample derived from ahost are well known to those of skill in the art. Such assay methodsinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis and ELISA assays.

Assaying TR21 and TR22 protein levels in a biological sample can occurusing any art-known method. By “biological sample” is intended anybiological sample obtained from an individual, cell line, tissueculture, or other source containing TR21 or TR22 receptor protein ormRNA. Preferred for assaying TR21 and TR22 protein levels in abiological sample are antibody-based techniques. For example, TR21 orTR22 protein expression in tissues can be studied with classicalimmunohistological methods. (M. Jalkanen et al., J Cell. Biol.101:976-985 (1985); M. Jalkanen et al., J Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting TR21 and TR22receptor gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA).

Suitable antibody assay labels are known in the art and include enzymelabels, such as, glucose oxidase; radioisotopes, such as iodine (¹³¹I,¹²⁵I, ¹²³I, ¹²¹I), carbon (14C), sulfur (35S), tritium (³H), indium (limIn, 113mIn, 112In, 1 In), and technetium (⁹⁹Tc, ⁹⁹mTc), thallium(²⁰Ti), gallium (Ga, ⁶⁷Ga), palladium (1⁰³Pd), molybdenum (99Mo), xenon(1³³Xe), fluorine (¹⁸F), 1⁵³sm, ¹⁷⁷Lu, ¹⁵⁹Gd, 1⁴⁹Pm, 1⁴⁰La, 175n, ¹⁶⁶1⁸⁸Re, 1⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin.Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which immunospecifically bind a polypeptide, preferablyan epitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGI,IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab and F(ab)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CHI, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CHi, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, shiprabbit, goat, guinea pig, camel, horse, or chicken. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins,as described infra and, for example in, U.S. Patent No. 5,939,598 byKucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention that they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies thatspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same. 10196] Antibodies of thepresent invention may also be described or specified in terms of theircross-reactivity. Antibodies that do not bind any other analog,ortholog, or homolog of a polypeptide of the present invention areincluded. Antibodies that bind polypeptides with at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least65%, at least 60%, at least 55%, and at least 50% identity (ascalculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. Antibodies that do not bind polypeptides with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. Further included in the present invention areantibodies that bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5X10-²M, 10-2M, 5X10-M, 10-³M, 5X10⁴M, 1010-⁵M, 5X10-⁶M, 10-⁶M, 5X10-⁷M,10-⁷M, 5X10-⁸M, 10-M, 5X1O-M, 10-M, 5X102M, 10-12M, 5X10-M, 10-M,5X1O-145XI0-¹⁵M, and 10-1⁵M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand or receptor activity by at least 90%,at least 80%, at least 70%, at least 60%, or at least 50% of theactivity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation. The antibodiesmay be specified as agonists, antagonists or inverse agonists forbiological activities comprising the specific biological activities ofthe peptides of the invention disclosed herein. The above antibodyagonists can be made using methods known in the art. See, e.g., PCTpublication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood92(6):1981-1988 (1998); Chen, et al., Cancer Res. 58(16):3668-3678(1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al.,Cancer Res. 58(15):3209- 3214 (1998); Yoon, et al., J. Immunol.160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. I1I(Pt2):237-247(1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997);Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol.Chem. 272(17):11295-11301 (1997); Tarymnan et al., Neuron 14(4):755-762(1995); Muller et Structure 6(9):1153-1167 (1998); Bartunek et al.,Cytokine 8(l):14-20 (1996) (which are all incorporated by referenceherein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety). 102011 As discussed in more detail below, theantibodies of the present invention may be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S.Patent No. 5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e, by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art.For example, a polypeptide of the invention can be administered tovarious host animals including, but not limited to, rabbits, mice, rats,etc. to induce the production of sera containing polyclonal antibodiesspecific for the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. 102051 Methodsfor producing and screening for specific antibodies using hybridomatechnology are routine and well-known in the art and are discussed indetail in Example 6, below. Briefly, mice can be immnunized with apolypeptide of the invention or a cell expressing such peptide. Once animmune response is detected, e.g., antibodies specific for the antigenare detected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the ATCC. Hybridomas are selected and cloned bylimited dilution. The hybridoma clones are then assayed by methods knownin the art for cells that secrete antibodies capable of binding apolypeptide of the invention. Ascites fluid, which generally containshigh levels of antibodies, can be generated by immunizing mice withpositive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Imnunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entireties. Humanized antibodies areantibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat.Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as Abgenix, Inc. (Freemont,Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. lnnunol. 147(8):2429-2438 (1991)). For exampleantibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofFIGS. 1A-B or FIG. 2.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be obtained from a suitable source (e.g., an antibodycDNA library, or a cDNA library generated from, or nucleic acid,preferably poly A+ RNA, isolated from, any tissue or cells expressingthe antibody, such as hybridoma cells selected to express an antibody ofthe invention) by PCR amplification using synthetic primers hybridizableto the 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties ), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423- 42;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-54) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,1988, Science 242:1038- 1041).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, e.g., a heavy or light chain of anantibody of the invention, requires construction of an expression vectorcontaining a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule or a heavy or light chainof an antibody, or portion thereof (preferably containing the heavy orlight chain variable domain), of the invention has been obtained, thevector for the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the invention, or a heavy orlight chain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non- essential region of the viral genome (e.g., regionE1 or E3) will result in a recombinant virus that is viable and capableof expressing the antibody molecule in infected hosts. (e.g., see Logan& Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalski &Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro,which confers resistance to hygromycin (Santerre et al., 1984, Gene30:147). Methods commonly known in the art of recombinant DNA technologywhich can be used are described in Ausubel et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150: 1, which are incorporated by referenceherein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol.3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isampliflable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

Antibody Cojugates

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20 or 50 amino acids of the polypeptide) of the present invention togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. The antibodies may bespecific for antigens other than polypeptides (or portion thereof,preferably at least 10, 20 or 50 amino acids of the polypeptide) of thepresent invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337- 11341(1992) (said references incorporated by reference intheir entireties).

As discussed, supra, the polypeptides of the present invention may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides of the presentinvention may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995)0.

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitates theirpurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. See,for example, U.S. Pat. No. 4,741,900 for metal ions which can beconjugated to antibodies for use as diagnostics according to the presentinvention. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), andtechnetium (⁹⁹Tc, ⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F),¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion. A cytotoxin orcytotoxic agent includes any agent that is detrimental to cells.Examples include paclitaxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 40° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C, washing the beads in lysis buffer and resuspendingthe beads in SDS/sample buffer. The ability of the antibody of interestto immunoprecipitate a particular antigen can be assessed by, e.g.,western blot analysis. One of skill in the art would be knowledgeable asto the parameters that can be modified to increase the binding of theantibody to an antigen and decrease the background (e.g., pre-clearingthe cell lysate with sepharose beads). For further discussion regardingimmunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%- 20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest is conjugated to a labeled compound (e.g., 3H or125I) in the presence of increasing amounts of an unlabeled secondantibody.

Antibody-Based Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the described disorders. Therapeutic compounds of theinvention include, but are not limited to, antibodies of the invention(including fragments, analogs and derivatives thereof as describedherein) and nucleic acids encoding antibodies of the invention(including fragments, analogs and derivatives thereof as describedherein). The antibodies of the invention can be used to treat, inhibitor prevent diseases and disorders associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, immune disorders. The treatment and/or prevention ofdiseases and disorders associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases and disorders.Antibodies of the invention may be provided in pharmaceuticallyacceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragments thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10−6 M, 10−6 M, 5×10−7 M,10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 m, 5×10−11M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M,5×10−15 M, and 10−15 M.

Antibody-Based Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody nucleic acids(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935;Zijlstra et al., 1989, Nature 342:435-438). In specific embodiments, theexpressed antibody molecule is a single chain antibody; alternatively,the nucleic acid sequences include sequences encoding both the heavy andlight chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992(Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993(Clarke et al.), WO 93/20221 dated Oct. 14, 1993 (Young)).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., 1993, Meth. Enzymol.217:581-599). These retroviral vectors have been to delete retroviralsequences that are not necessary for packaging of the viral genome andintegration into host cell DNA. The nucleic acid sequences encoding theantibody to be used in gene therapy are cloned into one or more vectors,which facilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., 1994, Biotherapy6:291-302, which describes the use of a retroviral vector to deliver themdrl gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin.Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons andGunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson,1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143- 155;Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang, et al., 1995, Gene Therapy 2:775-783. In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300;U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598, dated Apr. 28,1994; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980,Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Demonstration of Antibody-Based Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Antibody-Based Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction include butare not limited to intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes. Thecompounds or compositions may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceutical compoundsor compositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, 1990, Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp.353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201;Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J.Med. 321:574). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem.23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989,Ann. Neurol. 25:351; Howard et al., 1989, J.Neurosurg. 71:105). In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E.W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration. 102821 In apreferred embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration. [02831 The compounds of the invention can be formulatedas neutral or salt forms. Pharmaceutically acceptable salts includethose formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc. [0284] The amount of the compound of theinvention which will be effective in the treatment, inhibition andprevention of a disease or disorder associated with aberrant expressionand/or activity of a polypeptide of the invention can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.[0285] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation. [0286] The invention also provides a pharmaceuticalpack or kit comprising one or more containers filled with one or more ofthe ingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. Antibody-Based Diagnosis and Imaging [0287] Labeledantibodies, and derivatives and analogs thereof, which specifically bindto a polypeptide of interest can be used for diagnostic purposes todetect, diagnose, or monitor diseases and/or disorders associated withthe aberrant expression and/or activity of a polypeptide of theinvention. The invention provides for the detection of aberrantexpression of a polypeptide of interest, comprising (a) assaying theexpression of the polypeptide of interest in cells or body fluid of anindividual using one or more antibodies specific to the polypeptideinterest and (b) comparing the level of gene expression with a standardgene expression level, whereby an increase or decrease in the assayedpolypeptide gene expression level compared to the standard expressionlevel is indicative of aberrant expression. [02881 The inventionprovides a diagnostic assay for diagnosising a disorder, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of a particular disorder. With respect tocancer, the presence of a relatively high amount of transcript inbiopsied tissue from an individual may indicate a predisposition for thedevelopment of the disease, or may provide a means for detecting thedisease prior to the appearance of actual clinical symptoms. A moredefinitive diagnosis of this type may allow health professionals toemploy preventative measures or aggressive treatment earlier therebypreventing the development or further progression of the cancer. [02891Antibodies of the invention can be used to assay protein levels in abiological sanple using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, M., et al., J. Cell.Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol.105:3087-3096 (1987)). Other antibody-based methods useful for detectingprotein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase; radioisotopes, such as iodine (I, I251, I21,I)0, carbon (¹⁴C), sulfur (35S), tritium (3 H), indium (5mIn, “In, 12In,In), and technetium (⁹⁹Tc, ⁹⁹mTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga,⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine(1⁸F), ¹⁵³sm, ¹⁷⁷Lu, ¹⁵⁹Gd, 1⁴⁹Pm, La, 175, 1⁶⁶Ho, ⁹⁰y, ⁴⁷Sc, ¹⁸⁶Re,88Re, 42Pr, 105Rh, ⁹⁷Ru; luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin.[02901 One aspect of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof the interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled molecule in the subject, such that detection of labeled moleculeabove the background level indicates that the subject has a particulardisease or disorder associated with aberrant expression of thepolypeptide of interest. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system. [02911It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S.W. Burchiel et al., “Iimrnunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982). [02921 Depending on severalvariables, including the type of label used and the mode ofadministration, the time interval following the administration forpermitting the labeled molecule to preferentially concentrate at sitesin the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or S to 10 days. [0293] In an embodiment, monitoring of the diseaseor disorder is carried out by repeating the method for diagnosing thedisease or disease, for example, one month after initial diagnosis, sixmonths after initial diagnosis, one year after initial diagnosis, etc.[0294] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography. [0295] In a specificembodiment, the molecule is labeled with a radioisotope and is detectedin the patient using a radiation responsive surgical instrument(Thurston et al., U.S. Patent No. 5,441,050). In another embodiment, themolecule is labeled with a fluorescent compound and is detected in thepatient using a fluorescence responsive scanning instrument. In anotherembodiment, the molecule is labeled with a positron emitting metal andis detected in the patent using positron emission-tomography. In yetanother embodiment, the molecule is labeled with a paramagnetic labeland is detected in a patient using magnetic resonance imaging (MRI).Antibody-Based Kits [0296] The present invention provides kits that canbe used in the above methods. In one embodiment, a kit comprises anantibody of the invention, preferably a purified antibody, in one ormore containers. In a specific embodiment, the kits of the presentinvention contain a substantially isolated polypeptide comprising anepitope which is specifically immunoreactive with an antibody includedin the kit. Preferably, the kits of the present invention furthercomprise a control antibody which does not react with the polypeptide ofinterest. In another specific embodiment, the kits of the presentinvention contain a means for detecting the binding of an antibody to apolypeptide of interest (e.g., the antibody may be conjugated to adetectable substrate such as a fluorescent compound, an enzymaticsubstrate, a radioactive compound or a luminescent compound, or a secondantibody which recognizes the first antibody may be conjugated to adetectable substrate). [0297] In another specific embodiment of thepresent invention, the kit is a diagnostic kit for use in screeningserum containing antibodies specific against proliferative and/orcancerous polynucleotides and polypeptides. Such a kit may include acontrol antibody that does not react with the polypeptide of interest.Such a kit may include a substantially isolated polypeptide antigencomprising an epitope which is specifically immunoreactive with at leastone anti-polypeptide antigen antibody. Further, such a kit includesmeans for detecting the binding of said antibody to the antigen (e.g.,the antibody may be conjugated to a fluorescent compound such asfluorescein or rhodamine which can be detected by flow cytometry). Inspecific embodiments, the kit may include a recombinantly produced orchemically synthesized polypeptide antigen. The polypeptide antigen ofthe kit may also be attached to a solid support. 10298] In a morespecific embodiment the detecting means of the above-described kitincludes a solid support to which said polypeptide antigen is attached.Such a kit may also include a non-attached reporter-labeled anti-humanantibody. In this embodiment, binding of the antibody to the polypeptideantigen can be detected by binding of the said reporter- labeledantibody. 102991 In an additional embodiment, the invention includes adiagnostic kit for use in screening serum containing antigens of thepolypeptide of the invention. The diagnostic kit includes asubstantially isolated antibody specifically immunoreactive withpolypeptide or polynucleotide antigens, and means for detecting thebinding of the polynucleotide or polypeptide antigen to the antibody. hione embodiment, the antibody is attached to a solid support. In aspecific embodiment, the antibody may be a monoclonal antibody. Thedetecting means of the kit may include a second, labeled monoclonalantibody. Alternatively, or in addition, the detecting means may includea labeled, competing antigen. [0300] In one diagnostic configuration,test serum is reacted with a solid phase reagent having a surface-boundantigen obtained by the methods of the present invention. After bindingwith specific antigen antibody to the reagent and removing unbound serumcomponents by washing, the reagent is reacted with reporter-labeledanti-human antibody to bind reporter to the reagent in proportion to theamount of bound anti-antigen antibody on the solid support. The reagentis again washed to remove unbound labeled antibody, and the amount ofreporter associated with the reagent is determined. Typically, thereporter is an enzyme which is detected by incubating the solid phase inthe presence of a suitable fluorometric, luminescent or colorimetricsubstrate (Sigma, St. Louis, MO). [03011 The solid surface reagent inthe above assay is prepared by known techniques for attaching proteinmaterial to solid support material, such as polymeric beads, dip sticks,96-well plate or filter material. These attachment methods generallyinclude non- specific adsorption of the protein to the support orcovalent attachment of the protein, typically through a free aminegroup, to a chemically reactive group on the solid support, such as anactivated carboxyl, hydroxyl, or aldehyde group. Alternatively,streptavidin coated plates can be used in conjunction with biotinylatedantigen(s). [03021 Thus, the invention provides an assay system or kitfor carrying out this diagnostic method. The kit generally includes asupport with surface- bound recombinant antigens, and a reporter-labeledanti-human antibody for detecting surface-bound anti- antigen antibody.Therapeutic Compositions and Methods [03031 As demonstrated in FIG. 7,transient expression of transfected TR21 DNA in 293T cells, in thepresence of a NF-kB-SEAP reporter construct, activated the NF-KBtranscription complex in a dose dependent manner, indicating that TR21plays a role as a cellular proliferative factor. Accordingly, inpreferred embodiments, TR21 and TR22 polynucleotides, polypeptides andagonistic antibodies of the invention are useful as cellularproliferative factors in disorders requiring enhanced proliferation ofparticular cell types, such as for example, tissues demonstratingexpression of TR21 and TR22, as discussed supra. [0304] Alternatively,TR21 and TR22 antagonists, including antagonistic antibodies, are usefulfor the prevention and/or inhibition of the proliferation of particularcell types in diseases requiring a decrease in cellular proliferation,such as for example, tissues demonstrating expression of TR21 and TR22,as discussed supra. [03051 The Tumor Necrosis Factor (TNF) familyligands are known to be among the most pleiotropic cytokines, inducing alarge number of cellular responses, including cytotoxicity, anti-viralactivity, immunoregulatory activities, and the transcriptionalregulation of several genes (D.V. Goeddel et al., “Tumor NecrosisFactors: Gene Structure and Biological Activities,” Symp. Quant. Biol.51:597- 609 (1986), Cold Spring Harbor; B. Beutler and A. Cerami, Annu.Rev. Biochem. 57:505-518 (1988); L.J. Old, Sci. Am. 258:59-75 (1988); W.Fiers, FEBS Lett. 285:199-224 (1991)). The TNF-family ligands inducesuch various cellular responses by binding to TNF-family receptors,including the TR21 and TR22 of the present invention. [0306] TR21 andTR22 polynucleotides, polypeptides, agonists and/or antagonists of theinvention may be administered to a patient (e.g., mammal, preferablyhuman) afflicted with any disease or disorder mediated (directly orindirectly) by defective, or deficient levels of, TR21 or TR22.Alternatively, a gene therapy approach may be applied to treat suchdiseases or disorders. In one embodiment of the invention, TR21 or TR22polynucleotide sequences are used to detect mutein TR21 or TR22 genes,including defective genes. Mutein genes may be identified in in vitrodiagnostic assays, and by comparison of the TR21 or TR22 nucleotidesequence disclosed herein with that of a TR21 or TR22 gene obtained froma patient suspected of harboring a defect in this gene. Defective genesmay be replaced with normal TR21 or TR22-encoding genes using techniquesknown to one skilled in the art. [03071 In another embodiment, the TR21or TR22 polypeptides, polynucleotides, agonists and/or antagonists ofthe present invention are used as research tools for studying thephenotypic effects that result from inhibiting Ligand/TR21 Ligan/TR22interactions on various cell types. TR21 and TR22 polypeptides andantagonists (e.g. monoclonal antibodies to TR21 or TR22) also may beused in in vitro assays for detecting ligand, TR2 1, or TR22 or theinteractions thereof. [03081 The therapeutic compositions and methodsdescribed in this section include those antibody-based composition andmethods described in detail above. For example, the agonists andantagonists, and methods of using such agonists and antagoists, includethe antibodies and their uses described above. [0309] Cells whichexpress the TR21 and TR22 polypeptide and are believed to have a potentcellular response to TR21 and TR22 ligands include fetal liver, PBL,lung, kidney, small intestine, colon, keratinocytes, endothelial cells,and monocyte activated tissue. By “a cellular response to a TNF-familyligand” is intended any genotypic, phenotypic, and/or morphologic changeto a cell, cell line, tissue, tissue culture or patient that is inducedby a TNF-family ligand. As indicated, such cellular responses includenot only normal physiological responses to TNF-family ligands, but alsodiseases associated with increased apoptosis or the inhibition ofapoptosis. Apoptosis-programmed cell death-is a physiological mechanisminvolved in the deletion of peripheral T lymphocytes of the immunesystem, and its dysregulation can lead to a number of differentpathogenic processes (J.C. Ameisen, AIDS 8:1197-1213 (1994); P.H.Krammer et al., Curr. Opin. Immunol. 6:279-289 (1994)). [0310] Diseasesassociated with increased cell survival, or the inhibition of apoptosis,and that may be treated or prevented by the polynucleotides,polypeptides and/or agonists or antagonists of the invention include,but are not limited to, cancers (such as follicular lymphomas,carcinomas with p53 mutations, and hormone-dependent tumors, including,but not limited to colon cancer, cardiac tumors, pancreatic cancer,melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostrate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis rheumatoidarthritis); viral infections (such as herpes viruses, pox viruses andadenoviruses); inflammation; graft vs. host disease; acute graftrejection and chronic graft rejection. In preferred embodiments, TR21 orTR22 polynucleotides, polypeptides, and/or antagonists of the inventionare used to inhibit growth, progression, and/or metasis of cancers, inparticular those listed above, or in the paragraph that follows. [03111Additional diseases or conditions associated with increased cellsurvival and that may be treated or prevented by the polynucleotides,polypeptides and/or agonists or antagonists of the invention include,but are not limited to, progression, and/or metastases of malignanciesand related disorders such as leukemia (including acute leukemias (e.g.,acute lymphocytic leukemia, acute myelocytic leukemia (includingmyeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma. [0312] Thus, in preferredembodiments TR21 or TR22 polynucleotides or polypeptides of theinvention and agonists or antagonists thereof, are used to treat orprevent autoimmune diseases and/or inhibit the growth, progression,and/or metastasis of cancers, including, but not limited to, thosecancers disclosed herein, such as, for example, lymphocytic leukemias(including, for example, MLL and chronic lymphocytic leukemia (CLL)) andfollicular lymphomas. In another embodiment TR21 or TR22 polynucleotidesor polypeptides of the invention and/or agonists or antagonists thereof,are used to activate, differentiate or proliferate cancerous cells ortissue (e.g., B cell lineage related cancers (e.g., CLL and MLL),lymphocytic leukemia, or lymphoma) and thereby render the cells morevulnerable to cancer therapy (e.g., chemotherapy or radiation therapy).10313] Diseases associated with increased apoptosis and that may betreated or prevented by the polynucleotides, polypeptides and/oragonists or antagonists of the invention include, but are not limitedto, AIDS; neurodegenerative disorders (such as Alzheimer's disease,Parkinson's disease, Amyotrophic lateral sclerosis, Retinitispiginentosa, Cerebellar degeneration and brain tumor or prior associateddisease); autoimmune disorders (such as, multiple sclerosis, Sjogren'ssyndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease,Crohn's disease, polymyositis, systemic lupus erythematosus andimmune-related glomerulonephritis and rheumatoid arthritis);myelodysplastic syndromes (such as aplastic anemia), graft v. hostdisease, ischemic injury (such as that caused by myocardial infarction,stroke and reperfusion injury), liver injury (such as hepatitis relatedliver injury, ischemia/reperfusion injury, cholestosis (bile ductinjury) and liver cancer); toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia and anorexia. In preferredembodiments, TR21 or TR22 polynucleotides, polypeptides and/or agonistsare used to treat the diseases and disorders listed above. [0314] Manyof the pathologies associated with HIV are mediated by apoptosis,including HIV-induced nephropathy and HIV encephalitis. Thus, inadditional preferred embodiments, TR21 or TR22 polynucleotides,polypeptides, and/or TR21 or TR22 agonists or antagonists of theinvention are used to treat AIDS and pathologies associated with AIDS.[03151 Another embodiment of the present invention is directed to theuse of TR21 or TR22 to reduce death of T cells in HIV-infected patients.The state of immunodeficiency that defines AIDS is secondary to adecrease in the number and function of CD4+T- lymphocytes. Recentreports estimate the daily loss of CD4+T cells to be between 3.5 x 107and 2 x 109 cells (Wei et al., Nature 373:117-122 (1995)). One cause ofCD4+T cell depletion in the setting of HIV infection is believed to beHIV-induced apoptosis (see, for example, Meyaard et al., Science257:217-219, 1992; Groux et al., JExp. Med., 175:331, 1992; and Oyaizuet al., in Cell Activation and Apoptosis in HIV Infection, Andrieu andLu, Eds., Plenum Press, New York, 1995, pp. 101-114). Indeed,HIV-induced apoptotic cell death has been demonstrated not only in vitrobut also, more importantly, in infected individuals (J.C. Ameisen, AIDS8:1197-1213 (1994); T.H. Finkel and N.K. Banda, Curr. Opin. Immunol.6:605-615(1995); C.A. Muro-Cacho et al., J. Immunol. 154:5555-5566(1995)). Furthermore, apoptosis and CD4+T-lymphocyte depletion istightly correlated in different animal models of AIDS (T. Brunner etal., Nature 373:441-444 (1995); M.L. Gougeon et al., AIDS Res. Hum.Retroviruses 9:553-563 (1993)) and, apoptosis is not observed in thoseanimal models in which viral replication does not result in AIDS. Id.Further data indicates that uninfected but primed or activated Tlymphocytes from HIV- infected individuals undergo apoptosis afterencountering the TNF-family ligand FasL. Using monocytic cell lines thatresult in death following HIV infection, it has been demonstrated thatinfection of U937 cells with HIV results in the de novo expression ofFasL and that FasL mediates HIV-induced apoptosis (A.D. Badley et al., JVirol. 70:199- 206 (1996)). Further, the TNF-family ligand wasdetectable in uninfected macrophages and its expression was upregulatedfollowing HIV infection resulting in selective killing of uninfected CD4T-lymphocytes. Id. Thus, by the invention, a method for treatingHIV+individuals is provided which involves administering TR21 or TR22and/or TR21 or TR22 agonists of the present invention to reduceselective killing of CD4+T-lymphocytes. Modes of administration anddosages are discussed in detail below. [0316] Activated human T cellsare induced to undergo programmed cell death (apoptosis) upon triggeringthrough the CD3/T cell receptor complex, a process termedactivated-induced cell death (AICD). AICD of CD4+T cells isolated fromHIV-Infected asymptomatic individuals has been reported (Groux et al.,supra). Thus, AICD may play a role in the depletion of CD4+T cells andthe progression to AIDS in HIV-infected individuals. Thus, the presentinvention provides a method of inhibiting TRAIL-mediated T cell death inHIV patients, comprising administering a TR21 or TR22 polypeptide of theinvention (preferably, a soluble TR21 or TR22 polypeptide) to thepatients. In one embodiment, the patient is asymptomatic when treatmentwith TR21 or TR22 commences. If desired, prior to treatment, peripheralblood T cells may be extracted from an HIV patient, and tested forsusceptibility to ligand-mediated cell death by procedures known in theart. In one embodiment, a patient's blood or plasma is contacted withTR21 or TR22 ex vivo. The TR21 or TR22 may be bound to a suitablechromatography matrix by procedures known in the art. The patient'sblood or plasma flows through a chromatography column containing TR21 orTR22 bound to the matrix, before being returned to the patient. Theimmobilized TR21 or TR22 binds ligand, thus removing the ligand from thepatient's blood. [0317] In additional embodiments a TR21 or TR22polypeptide of the invention is administered in combination with otherinhibitors of T cell apoptosis. For example, Fas-mediated apoptosis alsohas been implicated in loss of T cells in HIV individuals (Katsikis etal., J. Exp. Med. 181:2029-2036 (1995)). Thus, a patient susceptible toFas ligand mediated, or TRAIL mediated T cell death, or both, forexample may be treated with both an agent that blocks TRAIL/TRAILreceptor interactions and/or an agent that blocks Fas-ligand/Fasinteractions. Suitable agents for blocking binding of Fas-ligand to Fasinclude, but are not limited to, soluble Fas polypeptides; mulitmericforms of soluble Fas polypeptides (e.g., dimers of sFas/Fc); anti-Fasantibodies that bind Fas without transducing the biological signal thatresults in apoptosis; anti-Fas-ligand antibodies that block binding ofFas-ligand to Fas; and muteins of Fas-ligand that bind Fas but do nottransduce the biological signal that results in apoptosis. Preferably,the antibodies employed according to this method are monoclonalantibodies. Examples of suitable agents for blocking Fas-ligand/Fasinteractions, including blocking anti-Fas monoclonal antibodies, aredescribed in International application publication number WO 95/10540,hereby incorporated by reference. [03181 Suitable agents, which blockbinding of TRAIL to a TRAIL receptor that may be administered with thepolynucleotides and/or polypeptides of the present invention include,but are not limited to, soluble TRAIL receptor polypeptides (e.g., asoluble form of OPG, DR4 (International application publication numberWO 98/32856); TR5 (International application publication number WO98/30693); and DR5 (International application publication number WO98/41629)); multimeric forms of soluble TRAIL receptor polypeptides; andTRAIL receptor antibodies that bind the TRAIL receptor withouttransducing the biological signal that results in apoptosis, anti-TRAILantibodies that block binding of TRAIL to one or more TRAIL receptors,and muteins of TRAIL that bind TRAIL receptors but do not transduce thebiological signal that results in apoptosis. Preferably, the antibodiesemployed according to this method are monoclonal antibodies. [0319] TR21andTR22 polypeptides or polynucleotides encoding TR21 and TR22 of theinvention may be used to treat cardiovascular disorders, includingperipheral artery disease, such as limb ischemia. [0320] Cardiovasculardisorders include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenousmalformations, congenital heart defects, pulmonary atresia, and ScimitarSyndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia,patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex,hypoplastic left heart syndrome, levocardia, tetralogy of fallot,transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, suchas aortopulmonary septal defect, endocardial cushion defects,Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septaldefects, and conditions characterized by clotting of small bloodvessels. [0321] Cardiovascular disorders also include heart disease,such as arrhythmias, carcinoid heart disease, high cardiac output, lowcardiac output, cardiac tamponade, endocarditis (including bacterial),heart aneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis. [03221Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre- excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.103231 Heart valve disease include aortic valve insufficiency, aorticvalve stenosis, hear murmurs, aortic valve prolapse, mitral valveprolapse, tricuspid valve prolapse, mitral valve insufficiency, mitralvalve stenosis, pulmonary atresia, pulmonary valve insufficiency,pulmonary valve stenosis, tricuspid atresia, tricuspid valveinsufficiency, and tricuspid valve stenosis. [0324] Myocardial diseasesinclude alcoholic cardiomyopathy, congestive cardiomyopathy,hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonarysubvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy,endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome,myocardial reperfusion injury, and myocarditis. 10325] Myocardialischemias include coronary disease, such as angina pectoris, coronaryaneurysm, coronary arteriosclerosis, coronary thrombosis, coronaryvasospasm, myocardial infarction and myocardial stunning. f03261Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel- Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno- occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,thrombotic microangiopathies (e.g., thrombotic thrombocytopenic purpura(TTP) and hemolytic-uremic syndrome (HUS)), and venous insufficiency.10327] Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms. 10328]Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans. [03291 Cerebrovasculardisorders include carotid artery diseases, cerebral amyloid angiopathy,cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebralarteriovenous malformation, cerebral artery diseases, cerebral embolismand thrombosis, carotid artery thrombosis, sinus thrombosis,Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subduralhematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebralischemia (including transient), subclavian steal syndrome,periventricular leukomalacia, vascular headache, cluster headache,migraine, and vertebrobasilar insufficiency. [0330] Embolisms includeair embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toesyndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms.Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinalvein occlusion, carotid artery thrombosis, sinus thrombosis,Wallenberg's syndrome, and thrombophlebitis. [03311 Ischemia includescerebral ischemia, ischemic colitis, compartment syndromes, anteriorcompartment syndrome, myocardial ischemia, reperfusion injuries, andperipheral limb ischemia. Vasculitis includes aortitis, arteritis,Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph nodesyndrome, thromboangiitis obliterans, hypersensitivity vasculitis,Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener'sgranulomatosis. [0332] The naturally occurring balance betweenendogenous stimulators and inhibitors of angiogenesis is one in whichinhibitory influences predominate. Rastinejad et al., Cell 56:345-355(1989). In those rare instances in which neovascularization occurs undernormal physiological conditions, such as wound healing, organregeneration, embryonic development, and female reproductive processes,angiogenesis is stringently regulated and spatially and temporallydelimited. Under conditions of pathological angiogenesis such as thatcharacterizing solid tumor growth, these regulatory controls fail.Unregulated angiogenesis becomes pathologic and sustains progression ofmany neoplastic and non- neoplastic diseases. A number of seriousdiseases are dominated by abnormal neovascularization including solidtumor growth and metastases, arthritis, some types of eye disorders, andpsoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634(1991); Folkman et al., N. Engl. J Med., 333:1757-1763 (1995); Auerbachet al., J Microvasc. Res. 29:401-411 (1985); Folkman, Advances in CancerResearch, eds. Klein and Weinhouse, Academic Press, New York, pp.175-203 (1985); Patz, Am. J Opthalmol. 94:715-743 (1982); and Folkman etal., Science 221:719-725 (1983). In a number of pathological conditions,the process of angiogenesis contributes to the disease state. Forexample, significant data have accumulated which suggest that the growthof solid tumors is dependent on angiogenesis. Folkman and Klagsbrun,Science 235:442-447 (1987). [0333] The present invention provides fortreatment of diseases or disorders associated with neovascularization byadministration of the TR21 or TR22 polynucleotides and/or polypeptidesof the invention (including TR21 or TR22 agonists and/or antagonists).Malignant and metastatic conditions which can be treated with thepolynucleotides and polypeptides of the invention include, but are notlimited to those malignancies, solid tumors, and cancers describedherein and otherwise known in the art (for a review of such disorders,see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia(1985)). (0334] Additionally, ocular disorders associated withneovascularization which can be treated with the TR21 or TR22polynucleotides and polypeptides of the present invention (includingTR21 or TR22 agonists and TR21 or TR22 antagonists) include, but are notlimited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma,retrolental fibroplasia, uveitis, retinopathy of prematurity maculardegeneration, corneal graft neovascularization, as well as other eyeinflammatory diseases, ocular tumors and diseases associated withchoroidal or iris neovascularization. See, e.g., reviews by Waltman etal., Am. J OphthaL 85:704-710 (1978) and Gartneretal., Surv. Ophthal.22:291-312 (1978). [0335] Additionally, disorders which can be treatedwith the TR21 or TR22 polynucleotides and polypeptides of the presentinvention (including TR21 or TR22 agonists and TR21 or TR22 antagonists)include, but are not limited to, hemangioma, arthritis, psoriasis,angiofibroma, atherosclerotic plaques, delayed wound healing,granulations, hemophilic joints, hypertrophic scars, nonunion fractures,Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, andvascular adhesions. [0336] The polynucleotides and/or polypeptides ofthe invention and/or agonists and/or antagonists thereof, can also beemployed to inhibit the proliferation and differentiation ofhematopoietic cells and therefore may be employed to protect bone marrowstem cells from chemotherapeutic agents during chemotherapy. Thisantiproliferative effect may allow administration of higher doses ofchemotherapeutic agents and, therefore, more effective chemotherapeutictreatment. 10337] The polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof, may also beemployed for the expansion of immature hematopoeitic progenitor cells,for example, granulocytes, macrophages or monocytes (e.g., C-kit+,Sca-l+), by temporarily preventing their differentiation. These bonemarrow cells may be cultured in vitro. Thus, TR21 or TR22 may be usefulas a modulator of hematopoietic stem cells in vitro for the purpose ofbone marrow transplantation and/or gene therapy. Since stem cells arerare and are most usefuil for introducing genes into for gene therapy,TR21 or TR22 can be used to isolate enriched populations of stem cells.Stem cells can be enriched by culturing cells in the presence ofcytotoxins, such as 5-Fu, which kills rapidly dividing cells, where asthe stem cells will be protected by TR21 or TR22. These stem cells canbe returned to a bone marrow transplant patient or can then be used fortransfection of the desired gene for gene therapy. In addition, TR21 orTR22 can be injected into animals which results in the release of stemcells from the bone marrow of the animal into the peripheral blood.These stem cells can be isolated for the purpose of autologous bonemarrow transplantation or manipulation for gene therapy. After thepatient has finished chemotherapy or radiation treatment, the isolatedstem cells can be returned to the patient. [0338] In a specificembodiment, polynucleotides and/or polypeptides of the invention and/orangonists and/or antagonists thereof may be used to increase theconcentration of blood cells in individuals in need of such increase(i.e., in hematopoietin therapy). Conditions that may be ameliorated byadministering the compositions of the invention include, but are notlimited to, neutropenia, anemia, and thrombocytopenia. [03391 In aspecific embodiment, the polynucleotides and/or polypeptides of theinvention (and/or agonists or antagonists thereof) are used inerythropoietin therapy, which is directed toward supplementing theoxygen carrying capacity of blood. Polynucleotides and/or polypeptidesof the invention (and/or agonists or antagonists thereof) may be used totreat or prevent diseases or conditions in patients generally requiringblood transfusions, such as, for example, trauma victims, surgicalpatients, dialysis patients, and patients with a variety of bloodcomposition-affecting disorders, such as, for example, hemophilia,cystic fibrosis, pregnancy, menstrual disorders, early anemia ofprematurity, spinal cord injury, aging, various neoplastic diseasestates, and the like. Examples of patient conditions that requiresupplementation of the oxygen carrying capacity of blood and which arewithin the scope of this invention, include,but are not limited to:treatment of blood disorders characterized by low or defective red bloodcell production, anemia associated with chronic renal failure,stimulation of reticulocyte response, development of ferrokineticeffects (such as plasma iron turnover effects and marrow transit timeeffects), erythrocyte mass changes, stimulation of hemoglobin Csynthesis, and increasing levels of hematocrit in vertebrates. Theinvention also provides for treatment to enhance the oxygen-carryingcapacity of an individual, such as for example, an individualencountering hypoxic environmental conditions. [03401 Polynucleotidesand/or polypeptides of the invention and/or angonists and/or antagoniststhereof may be used in treatment of myeloid leukemias. [03411 TR21 orTR22 polynucleotides or polypeptides, or agonists of TR21 or TR22, canbe used in the treatment of infectious agents. For example, byincreasing the immune response, particularly increasing theproliferation and differentiation of B cells, infectious diseases may betreated. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, TR2 1 or TR22 polynucleotides or polypeptides, oragonists or antagonists of TR21 or TR22, may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.[03421 Viruses are one example of an infectious agent that can causedisease or symptoms that can be treated by TR21 or TR22 polynucleotidesor polypeptides, or agonists of TR21 or TR22. Examples of viruses,include, but are not limited to the following DNA and RNA viruses andviral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus,Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae,Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis),Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster),Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae),Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza),Papiloma virus, Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae(such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus),Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g.,Rubivirus). Viruses falling within these families can cause a variety ofdiseases or symptoms, including, but not limited to: arthritis,bronchiollitis, respiratory syncytial virus, encephalitis, eyeinfections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome,hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis,Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis,opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma,chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies,the common cold, Polio, leukemia, Rubella, sexually transmitteddiseases, skin diseases (e.g., Kaposi's, warts), and viremia. TR21 orTR22 polynucleotides or polypeptides, or agonists or antagonists of TR21or TR22, can be used to treat or detect any of these symptoms ordiseases. In specific embodiments, TR21 or TR22 polynucleotides,polypeptides, or agonists are used to treat: meningitis, Dengue, EBV,and/or hepatitis (e.g., hepatitis B). In an additional specificembodiment TR21 or TR22 polynucleotides, polypeptides, or agonists areused to treat patients nonresponsive to one or more other commerciallyavailable hepatitis vaccines. In a further specific embodiment, TR21 orTR22 polynucleotides, polypeptides, or agonists are used to treat AIDS.(03431 Similarly, bacterial or fungal agents that can cause disease orsymptoms and that can be treated by TR21 or TR22 polynucleotides orpolypeptides, or agonists or antagonists of TR21 or TR22, include, butnot limited to, the following Gram-Negative and Gram-positive bacteriaand bacterial families and fungi: Actinomycetales (e.g.,Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans,Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae,Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi,Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis,Cryptococcosis, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coliand Enterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella,Salmonella (e.g., Salmonella typhi, and Salmonella paratyphi), Serratia,Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis,Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae,Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseriameningitidis, Pasteurellacea Infections (e.g., Actinobacillus,Heamophilus (e.g., Heamophilus influenza type B), Pasteurella),Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp.,Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal (e.g.,Streptococcus pneumoniae and Group B Streptococcus). These bacterial orfungal families can cause the following diseases or symptoms, including,but not limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. TR21 or TR22 polynucleotides or polypeptides, oragonists or antagonists of TR21 or TR22, can be used to treat or detectany of these symptoms or diseases. In specific embodiments, TR21 or TR22polynucleotides, polypeptides, or agonists thereof are used to treat:tetanus, Diptheria, botulism, and/or meningitis type B. [0344] Moreover,parasitic agents causing disease or symptoms that can be treated by TR21or TR22 polynucleotides or polypeptides, or agonists of TR21 or TR22,include, but not limited to, the following families or class: Amebiasis,Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine,Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis,Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g.,Plasmodium virax, Plasmodium falciparium, Plasmodium malariae andPlasmodium ovale). These parasites can cause a variety of diseases orsymptoms, including, but not limited to: Scabies, Trombiculiasis, eyeinfections, intestinal disease (e.g., dysentery, giardiasis), liverdisease, lung disease, opportunistic infections (e.g., AIDS related),malaria, pregnancy complications, and toxoplasmosis. TR21 or TR22polynucleotides or polypeptides, or agonists or antagonists of TR21 orTR22, can be used to treat or detect any of these symptoms or diseases.In specific embodiments, TR21 or TR22 polynucleotides, polypeptides, oragonists thereof are used to treat malaria. [0345] An additionalcondition, disease or symptom that can be treated by TR21 or TR22polynucleotides or polypeptides, or agonists of TR21 or TR22, isosteomyelitis. [0346] Preferably, treatment using TR21 or TR22polynucleotides or polypeptides, or agonists of TR21 or TR22, couldeither be by administering an effective amount of TR21 or TR22polypeptide to the patient, or by removing cells from the patient,supplying the cells with TR21 or TR22 polynucleotide, and returning theengineered cells to the patient (ex vivo therapy). Moreover, as furtherdiscussed herein, the TR21 or TR22 polypeptide or polynucleotide can beused as an adjuvant in a vaccine to raise an immune response againstinfectious disease. [03471 Additional preferred embodiments of theinvention include, but are not limited to, the use of TR21 or TR22polypeptides and functional agonists in the following applications:10348] Administration to an animal (e.g., mouse, rat, rabbit, hamster,guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep,dog, cat, non-human primate, and human, most preferably human) to boostthe immune system to produce increased quantities of one or moreantibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinityantibody production (e.g., IgG, IgA, IgM, and IgE), and/or to increasean immune response. [0349] Administration to an animal (including, butnot limited to, those listed above, and also including transgenicanimals) incapable of producing functional endogenous antibody moleculesor having an otherwise compromised endogenous immune system, but whichis capable of producing human immunoglobulin molecules by means of areconstituted or partially reconsituted immune system from anotheranimal (see, e.g., published PCT Application Nos. W098/24893,W0/9634096, WO/9633735, and W0/9110741. 10350] A vaccine adjuvant thatenhances immune responsiveness to specific antigen. [0351] An adjuvantto enhance tumor-specific immune responses. [0352] An adjuvant toenhance anti-viral immune responses. Anti-viral immune responses thatmay be enhanced using the compositions of the invention as an adjuvant,include virus and virus associated diseases or symptoms described hereinor otherwise known in the art. In specific embodiments, the compositionsof the invention are used as an adjuvant to enhance an immune responseto a virus, disease, or symptom selected from the group consisting of:AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). Inanother specific embodiment, the compositions of the invention are usedas an adjuvant to enhance an immune response to a virus, disease, orsymptom selected from the group consisting of: HIV/AIDS, Respiratorysyncytial virus, Dengue, Rotavirus, Japanese B encephalitis, Influenza Aand B, Parainfluenza, Measles, Cytomegalovirus, Rabies, Junin,Chikungunya, Rift Valley fever, Herpes simplex, and yellow fever. [0353]An adjuvant to enhance anti-bacterial or anti-fimgal immune responses.Anti- bacterial or anti-fimgal immune responses that may be enhancedusing the compositions of the invention as an adjuvant, include bacteriaor fungus and bacteria or fungus associated diseases or symptomsdescribed herein or otherwise known in the art. In specific embodiments,the compositions of the invention are used as an adjuvant to enhance animmune response to a bacteria or fungus, disease, or symptom selectedfrom the group consisting of: tetanus, Diphtheria, botulism, andmeningitis type B. In another specific embodiment, the compositions ofthe invention are used as an adjuvant to enhance an immune response to abacteria or fungus, disease, or symptom selected from the groupconsisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi,Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae,Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium(malaria). [0354] An adjuvant to enhance anti-parasitic immuneresponses. Anti-parasitic immune responses that may be enhanced usingthe compositions of the invention as an adjuvant, include parasite andparasite associated diseases or symptoms described herein or otherwiseknown in the art. In specific embodiments, the compositions of theinvention are used as an adjuvant to enhance an immune response to aparasite. In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response toPlasmodium (malaria). [0355] As a stimulator of B cell responsiveness topathogens. [0356] As an agent that elevates the immune status of anindividual prior to their receipt of immunosuppressive therapies. [03571As an agent to induce higher affinity antibodies. [0358] As an agent toincrease serum immunoglobulin concentrations. [0359] As an agent toaccelerate recovery of immunocompromised individuals. [0360] As an agentto boost immunoresponsiveness among aged populations. [0361] As animmune system enhancer prior to, during, or after bone marrow transplantand/or other transplants (e.g., allogeneic or xenogeneic organtransplantation). With respect to transplantation, compositions of theinvention may be administered prior to, concomitant with, and/or aftertransplantation. In a specific embodiment, compositions of the inventionare administered after transplantation, prior to the beginning ofrecovery of T-cell populations. In another specific embodiment,compositions of the invention are first administered aftertransplantation after the beginning of recovery of T cell populations,but prior to full recovery of B cell populations. 103621 As an agent toboost immunoresponsiveness among B cell immunodeficient individuals. Bcell immunodeficiencies that may be ameliorated or treated byadministering the TR21 or TR22 polypeptides or polynucleotides of theinvention, or agonists thereof, include, but are not limited to, SCID,congenital agammaglobulinemia, common variable immunodeficiency,Wiskott-Aldrich Syndrome, X-linked immunodeficiency with hyper IgM, andsevere combined immunodeficiency. [03631 As an agent to boostimmunoresponsiveness among individuals having an acquired loss of B cellfinction. Conditions resulting in an acquired loss of B cell functionthat may be ameliorated or treated by administering the TR21 Aor TR22polypeptides or polynucleotides of the invention, or agonists thereof,include, but are not limited to, HIV Infection, AIDS, bone marrowtransplant, and B cell chronic lymphocytic leukemia (CLL). 10364] As anagent to boost immunoresponsiveness among individuals having a temporaryimmune deficiency. Conditions resulting in a temporary immune deficiencythat may be ameliorated or treated by administering the TR21 or TR22polypeptides or polynucleotides of the invention, or agonists thereof,include, but are not limited to, recovery from viral infections (e.g.,influenza), conditions associated with malnutrition, recovery frominfectious mononucleosis, or conditions associated with stress, recoveryfrom measles, recovery from blood transfusion, recovery from surgery.[0365] As a regulator of antigen presentation by monocytes, dendriticcells, and/or B-cells. In one embodiment, TR21 or TR22 (in soluble,membrane-bound or transmembrane forms) enhances antigen presentation orantagonizes antigen presentation in vitro or in vivo. Moreover, inrelated embodiments, said enhancement or antagonization of antigenpresentation may be useful as an anti-tumor treatment or to modulate theimmune system. [0366] As an agent to direct an individuals immune systemtowards development of a humoral response (i.e. TH2) as opposed to a THIcellular response. [0367] As a means to induce tumor proliferation andthus make it more susceptible to anti-neoplastic agents. For example,multiple myeloma is a slowly dividing disease and is thus refractory tovirtually all anti-neoplastic regimens. If these cells were forced toproliferate more rapidly their susceptibility profile would likelychange. [03681 As a stimulator of B cell production in pathologies suchas AIDS, chronic lymphocyte disorder and/or Common VariableImmunodificiency; [0369] As a therapy for generation and/or regenerationof lymphoid tissues following surgery, trauma or genetic defect. [0370]As a gene-based therapy for genetically inherited disorders resulting inimmuno-incompetence such as observed among SCID patients. [0371] As anantigen for the generation of antibodies to inhibit or enhance TR21 orTR22 mediated responses. [0372] As a means of activating T cells. [0373]As pretreatment of bone marrow samples prior to transplant. Suchtreatment would increase B cell representation and thus acceleraterecover. [0374] As a means of regulating secreted cytokines that areelicited by TR21 or TR22. [0375] TR21 or TR22 polypeptides orpolynucleotides of the invention, or agonists may be used to modulateIgE concentrations in vitro or in vivo. [0376] Additionally, TR21 orTR22 polypeptides or polynucleotides of the invention, or agoniststhereof, may be used to treat or prevent IgE-mediated allergicreactions. Such allergic reactions include, but are not limited to,asthma, rhinitis, and eczema. [0377] All of the above describedapplications as they may apply to veterinary medicine. [0378]Antagonists of TR21 or TR22 include binding and/or inhibitoryantibodies, antisense nucleic acids, ribozymes or soluble forms of theTR21 or TR22 receptor(s) (e.g,. the TR21 and TR22-Fc molecule describedin Example 38). These would be expected to reverse many of theactivities of the ligand described above as well as find clinical orpractical application as: [0379] A means of blocking various aspects ofimmune responses to foreign agents or self. Examples include autoimmunedisorders such as lupus, and arthritis, as well as immunoresponsivenessto skin allergies, inflammation, bowel disease, injury and pathogens.[0380] A therapy for preventing the B cell proliferation and Igsecretion associated with autoimmune diseases such as idiopathicthrombocytopenic purpura, systemic lupus erythramatosus and MS. [03811An inhibitor of graft versus host disease or transplant rejection.[0382] A therapy for B cell malignancies such as ALL, Hodgkins disease,non- Hodgkins lymphoma, Chronic lymphocyte leukemia, plasmacytomas,multiple myeloma, Burkitt's lymphoma, and EBV-transformed diseases.[03831 A therapy for chronic hypergammaglobulinemeia evident in suchdiseases as monoclonalgammopathy of undetermined significance (MGUS),Waldenstrom's disease, related idiopathic monoclonalgammopathies, andplasmacytomas. [0384] A therapy for decreasing cellular proliferation ofLarge B-cell Lymphomas. [03851 A means of decreasing the involvement ofB cells and Ig associated with Chronic Myelogenous Leukemia. [03861 Animmunosuppressive agent(s). [03871 TR21 or TR22 polypeptides orpolynucleotides of the invention, or antagonists may be used to modulateIgE concentrations in vitro or in vivo. [03881 In another embodiment,administration of TR21 or TR22 polypeptides or polynucleotides of theinvention, or antagonists thereof, may be used to treat or preventIgE-mediated allergic reactions including, but not limited to, asthma,rhinitis, and eczema. [03891 The above-recited applications have uses ina wide variety of hosts. Such hosts include, but are not limited to,human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat,hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-humanprimate, and human. In specific embodiments, the host is a mouse,rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat.In preferred embodiments, the host is a mammal. In most preferredembodiments, the host is a human. [03901 The agonists and antagonistsmay be employed in a composition with a pharmaceutically acceptablecarrier, e.g., as described herein. [03911 Polynucleotides and/orpolypeptides of the invention and/or agonists and/or antagonists thereofare useful in the diagnosis and treatment or prevention of a wide rangeof diseases and/or conditions. Such diseases and conditions include, butare not limited to, cancer (e.g., immune cell related cancers, breastcancer, prostate cancer, ovarian cancer, follicular lymphoma, cancerassociated with mutation or alteration of p53, brain tumor, bladdercancer, uterocervical cancer, colon cancer, colorectal cancer, non-smallcell carcinoma of the lung, small cell carcinoma of the lung, stomachcancer, etc.), lymphoproliferative disorders (e.g., lymphadenopathy),microbial (e.g., viral, bacterial, etc.) infection (e.g., HIV-1infection, HIV-2 infection, herpesvirus infection (including, but notlimited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirusinfection, poxvirus infection, human papilloma virus infection,hepatitis infection (e.g., HAV, HBV, HCV, etc.), Helicobacter pyloriinfection, invasive Staphylococcia, etc.), parasitic infection,nephritis, bone disease (e.g., osteoporosis), atherosclerosis, pain,cardiovascular disorders (e.g., neovascularization, hypovascularizationor reduced circulation (e.g., ischemic disease (e.g., myocardialinfarction, stroke, etc.), AIDS, allergy, inflammation,neurodegenerative disease (e.g., Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, pigmentary retinitis, cerebellardegeneration, etc.), graft rejection (acute and chronic), graft vs. hostdisease, diseases due to osteomyelodysplasia (e.g., aplastic anemia,etc.), joint tissue destruction in rheumatism, liver disease (e.g.,acute and chronic hepatitis, liver injury, and cirrhosis), autoimmunedisease (e.g., multiple sclerosis, rheumatoid arthritis, systemic lupuserythematosus, immune complex glomerulonephritis, autoimmune diabetes,autoimmune thrombocytopenic purpura, Grave's disease, Hashimoto'sthyroiditis, etc.), cardiomyopathy (e.g., dilated cardiomyopathy),diabetes, diabetic complications (e.g., diabetic nephropathy, diabeticneuropathy, diabetic retinopathy), influenza, asthma, psoriasis,glomerulonephritis, septic shock, and ulcerative colitis. [0392]Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in promoting angiogenesis,regulating hematopoiesis and wound healing (e.g., wounds, burns, andbone fractures). [0393] Polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof are also useful asan adjuvant to enhance immune responsiveness to specific antigen,anti-viral immune responses. [0394] More generally, polynucleotidesand/or polypeptides of the invention and/or agonists and/or antagoniststhereof are useful in regulating (i.e., elevating or reducing) immuneresponse. For example, polynucleotides and/or polypeptides of theinvention may be useful in preparation or recovery from surgery, trauma,radiation therapy, chemotherapy, and transplantation, or may be used toboost immune response and/or recovery in the elderly andimmunocompromised individuals. Alternatively, polynucleotides and/orpolypeptides of the invention and/or agonists and/or antagonists thereofare useful as immunosuppressive agents, for example in the treatment orprevention of autoimmune disorders. hn specific embodiments,polynucleotides and/or polypeptides of the invention are used to treator prevent chronic inflammatory, allergic or autoimmune conditions, suchas those described herein or are otherwise known in the art. [03951 Inone aspect, the present invention is directed to a method for enhancingapoptosis induced by a TNF-family ligand, which involves administeringto a cell which expresses the TR21 or TR22 polypeptide an effectiveamount of TR21 or TR22 ligand, analog or an agonist capable ofincreasing TR21 or TR22 mediated signaling. Preferably, TR21 or TR22mediated signaling is increased to treat a disease wherein decreasedapoptosis or decreased cytokine and adhesion molecule expression isexhibited. An agonist can include soluble forms of TR21 or TR22 andmonoclonal antibodies directed against the TR21 or TR22 polypeptide.[03961 In a further aspect, the present invention is directed to amethod for inhibiting apoptosis induced by a TNF-family ligand, whichinvolves administering to a cell which expresses the TR21 or TR22polypeptide an effective amount of an antagonist capable of decreasingTR21 or TR22 mediated signaling. Preferably, TR21 or TR22 mediatedsignaling is decreased to treat a disease wherein increased apoptosis orNF-kB expression is exhibited. An antagonist can include soluble formsof TR21 or TR22 and monoclonal antibodies directed against the TR21 orTR22 polypeptide. (03971 By “agonist” is intended naturally occurringand synthetic compounds capable of enhancing or potentiating apoptosis.By “antagonist” is intended naturally occurring and synthetic compoundscapable of inhibiting apoptosis. Whether any candidate “agonist” or“antagonist” of the present invention can enhance or inhibit apoptosiscan be determined using art-known TNF-family ligand/receptor cellularresponse assays, including those described in more detail below. 103981One such screening procedure involves the use of melanophores which aretransfected to express the receptor of the present invention. Such ascreening technique is described in PCT WO 92/01810, published February6, 1992. Such an assay may be employed, for example, for screening for acompound which inhibits (or enhances) activation of the receptorpolypeptide of the present invention by contacting the melanophore cellswhich encode the receptor with both a TNF-family ligand and thecandidate antagonist (or agonist). Inhibition or enhancement of thesignal generated by the ligand indicates that the compound is anantagonist or agonist of the ligand/receptor signaling pathway. [0399]Other screening techniques include the use of cells which express thereceptor (for example, transfected CHO cells) in a system which measuresextracellular pH changes caused by receptor activation. For example,compounds may be contacted with a cell which expresses the receptorpolypeptide of the present invention and a second messenger response,e.g., signal transduction or pH changes, may be measured to determinewhether the potential compound activates or inhibits the receptor.104001 Another such screening technique involves introducing RNAencoding the receptor into Xenopus oocytes to transiently express thereceptor. The receptor oocytes may then be contacted with the receptorligand and a compound to be screened, followed by detection ofinhibition or activation of a calcium signal in the case of screeningfor compounds which are thought to inhibit activation of the receptor.[0401] Another screening technique well known in the art involvesexpressing in cells a construct wherein the receptor is linked to aphospholipase C or D. Exemplary cells include endothelial cells, smoothmuscle cells, embryonic kidney cells, etc. The screening may beaccomplished as hereinabove described by detecting activation of thereceptor or inhibition of activation of the receptor from thephospholipase signal. [0402] Another method involves screening forcompounds which inhibit activation of the receptor polypeptide of thepresent invention antagonists by determining inhibition of binding oflabeled ligand to cells which have the receptor on the surface thereof.Such a method involves transfecting a eukaryotic cell with DNA encodingthe receptor such that the cell expresses the receptor on its surfaceand contacting the cell with a compound in the presence of a labeledform of a known ligand. The ligand can be labeled, e.g., byradioactivity. The amount of labeled ligand bound to the receptors ismeasured, e.g., by measuring radioactivity of the receptors. If thecompound binds to the receptor as determined by a reduction of labeledligand which binds to the receptors, the binding of labeled ligand tothe receptor is inhibited. [0403] Further screening assays for agonistsand antagonists of the present invention are described in L.A.Tartagliaand D.V. Goeddel, J Biol. Chem. 267:4304-4307(1992). [0404] Thus, in afurther aspect, a screening method is provided for determining whether acandidate agonist or antagonist is capable of enhancing or inhibiting acellular response to a TNF-family ligand. The method involves contactingcells which express the TR21 or TR22 polypeptide with a candidatecompound and a TNF-family ligand, assaying a cellular response, andcomparing the cellular response to a standard cellular response, thestandard being assayed when contact is made with the ligand in absenceof the candidate compound, whereby an increased cellular response overthe standard indicates that the candidate compound is an agonist of theligand/receptor signaling pathway and a decreased cellular responsecompared to the standard indicates that the candidate compound is anantagonist of the ligand/receptor signaling pathway. By “assaying acellular response” is intended qualitatively or quantitatively measuringa cellular response to a candidate compound and/or a TNF-family ligand(e.g., determining or estimating an increase or decrease in T cellproliferation or tritiated thymidine labeling). By the invention, a cellexpressing the TR21 or TR22 polypeptide can be contacted with either anendogenous or exogenously administered TNF-family ligand. [0405]Antagonist according to the present invention include naturallyoccurring and synthetic compounds such as, for example, TNF familyligand peptide fragments, transforming growth factor, neurotransmitters(such as glutamate, dopamine, N- methyl-D- aspartate), tumor suppressors(p53), cytolytic T cells and antimetabolites. Preferred agonists includechemotherapeutic drugs such as, for example, cisplatin, doxorubicin,bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate andvincristine. Others include ethanol and -amyloid peptide. (Science267:1457-1458 (1995)). Further preferred agonists include polyclonal andmonoclonal antibodies raised against the TR21 or TR22 polypeptide, or afragment thereof. Such agonist antibodies raised against a TNF-familyreceptor are disclosed in L.A. Tartaglia et al., Proc. Natl. Acad. Sci.USA 88:9292-9296 (1991); and L.A.Tartaglia and D.V.Goeddel, J Biol.Chem. 267:4304- 4307(1992). See, also, PCT Application WO 94/09137.[0406] Agonists according to the present invention include naturallyoccurring and synthetic compounds such as, for example, the CD40 ligand,neutral amino acids, zinc, estrogen, androgens, viral genes (such asAdenovirus ElB, Baculovirus p35 and IAP, Cowpox virus crmA, Epstein-Barrvirus BHRF], LMP-1, African swine fever virus LMW5-HL, and Herpesvirusyl 34.5), calpain inhibitors, cysteine protease inhibitors, and tumorpromoters (such as PMA, Phenobarbital, and -Hexachlorocyclohexane).10407] Other potential antagonists include antisense molecules.Antisense technology can be used to control gene expression throughantisense DNA or RNA or through triple- helix formation. Antisensetechniques are discussed, for example, in Okano, J Neurochem. 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, FL (1988). Triple helix formation isdiscussed in, for instance Lee et al., Nucleic Acids Research 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA. [0408] In specificembodiments, antagonists according to the present invention are nucleicacids corresponding to the sequences contained in TR21 or TR22, or thecomplementary strand thereof, and/or to nucleotide sequences containedin the deposited clones HCFMV39 or HMUCLO1. In one embodiment, antisensesequence is generated internally by the organism, in another embodiment,the antisense sequence is separately administered (see, for example,Okano H. et al., J Neurochem. 56:560 (1991), and Oligodeoxynucleotidesas Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL(1988). Antisense technology can be used to control gene expressionthrough antisense DNA or RNA, or through triple-helix formation.Antisense techniques are discussed for example, in Okano, Neurochem.56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, FL (1988). Triple helix formation isdiscussed in, for instance, Lee et aL, Nucleic Acids Research 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA. [0409] For example, the 5′coding portion of a polynucleotide that encodes the mature polypeptideof the present invention may be used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription thereby preventing transcription and theproduction of the receptor. The antisense RNA oligonucleotide hybridizesto the MRNA in vivo and blocks translation of the MRNA molecule intoreceptor polypeptide. The oligonucleotides described above can also bedelivered to cells such that the antisense RNA or DNA may be expressedin vivo to inhibit production of the receptor. [04101 In one embodiment,the TR21 or TR22 antisense nucleic acid of the invention is producedintracellularly by transcription from an exogenous sequence. Forexample, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the TR21 or TR22 antisense nucleic acid.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others know inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding TR21 or TR22, or fragments thereof,can be by any promoter known in the art to act in vertebrate, preferablyhuman cells. Such promoters can be inducible or constitutive. Suchpromoters include, but are not limited to, the SV40 early promoterregion (Bernoist and Chambon, Nature 29:304-310 (1981), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al., Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner etal., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatorysequences of the metallothionein gene (Brinster, et al., Nature296:39-42 (1982)), etc. 10411] The antisense nucleic acids of theinvention comprise a sequence complementary to at least a portion of anRNA transcript of a TR21 or TR22 gene. However, absolutecomplementarity, although preferred, is not required. A sequence“complementary to at least a portion of an RNA,” referred to herein,means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case of doublestranded TR21 or TR22 antisense nucleic acids, a single strand of theduplex DNA may thus be tested, or triplex formation may be assayed. Theability to hybridize will depend on both the degree of complementarityand the length of the antisense nucleic acid Generally, the larger thehybridizing nucleic acid, the more base mismatches with a TR21 or TR22RNA it may contain and still form a stable duplex (or triplex as thecase may be). One skilled in the art can ascertain a tolerable degree ofmismatch by use of standard procedures to determine the melting point ofthe hybridized complex. [04121 Oligonucleotides that are complementaryto the 5′ end of the message, e.g., the 5′ untranslated sequence up toand including the AUG initiation codon, should work most efficiently atinhibiting translation. However, sequences complementary to the 3′untranslated sequences of mRNAs have been shown to be effective atinhibiting translation of mRNAs as well. See generally, Wagner, R.,Nature 372:333-335 (1994). Thus, oligonucleotides complementary toeither the 5′- or 3′- non- translated, non-coding regions of the TR21 orTR22 shown in Figures IA-B and 2, respectively, could be used in anantisense approach to inhibit translation of endogenous TR21 or TR22mRNA. Oligonucleotides complementary to the 5′ untranslated region ofthe MRNA should include the complement of the AUG start codon. Antisenseoligonucleotides complementary to MRNA coding regions are less efficientinhibitors of translation but could be used in accordance with theinvention. Whether designed to hybridize to the 5′-, 3′- or codingregion of TR21 or TR22 mRNA, antisense nucleic acids should be at leastsix nucleotides in length, and are preferably oligonucleotides rangingfrom 6 to about 50 nucleotides in length. In specific aspects theoligonucleotide is at least 10 nucleotides, at least 17 nucleotides, atleast 25 nucleotides or at least 50 nucleotides. [0413] Thepolynucleotides of the invention can be DNA or RNA or chimeric mixturesor derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl; Acad. Sci. U.S.A.86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. 84:648-652(1987); PCT Publication No. W088/09810, published December 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. W089/10134,published April 25, 1988), hybridization-triggered cleavage agents.(See, e.g., Krol et al., BioTechniques 6:958-976 (1988)) orintercalating agents. (See, e.g., Zon, Pharm. Res. 5:539-549 (1988)). Tothis end, the oligonucleotide may be conjugated to another molecule,e.g., a peptide, hybridization triggered cross-linking agent, transportagent, hybridization-triggered cleavage agent, etc. [04141 The antisenseoligonucleotide may comprise at least one modified base moiety which isselected from the group including, but not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, I-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl- 2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. [04151 The antisense oligonucleotide may alsocomprise at least one modified sugar moiety selected from the groupincluding, but not limited to, arabinose, 2-fluoroarabinose, xylulose,and hexose. [0416] In yet another embodiment, the antisenseoligonucleotide comprises at least one modified phosphate backboneselected from the group including, but not limited to, aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof. [04171 In yetanother embodiment, the antisense oligonucleotide is an a-anomericoligonucleotide. An ac-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual 0-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res. 15:6625-6641 (1987)). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)). [0418] Polynucleotides of the invention may besynthesized by standard methods known in the art, e.g. by use of anautomated DNA synthesizer (such as are commercially available fromBiosearch, Applied Biosystems, etc.). As examples, phosphorothioateoligonucleotides may be synthesized by the method of Stein et al. (Nucl.Acids Res. 16:3209 (1988)), methylphosphonate oligonucleotides can beprepared by use of controlled pore glass polymer supports (Sarin et al.,Proc. Natl. Acad. Sci. U.S.A. 85:7448- 7451 (1988)), etc. [0419] Whileantisense nucleotides complementary to the TR21 or TR22 coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred. [0420] Potential antagonistsaccording to the invention also include catalytic RNA, or a ribozyme(See, e.g., PCT International Publication WO 90/11364, published October4, 1990; Sarver et al, Science 247:1222-1225 (1990). While ribozymesthat cleave mRNA at site specific recognition sequences can be used todestroy TR21 or TR22 mRNAs, the use of hammerhead ribozymes ispreferred. Hammerhead ribozyrnes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within thenucleotide sequence of TR21 or TR22 (FIGS. 1A-B and 2, respectively).Preferably, the ribozyme is engineered so that the cleavage recognitionsite is located near the 5′ end of the TR21 or TR22 mRNA; i.e., toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts. [0421] As in the antisense approach,the ribozymes of the invention can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells which express TR21 or TR22 in vivo. DNAconstructs encoding the ribozyme may be introduced into the cell in thesame manner as described above for the introduction of antisenseencoding DNA. A preferred method of delivery involves using a DNAconstruct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol mH or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous TR21 or TR22 messages and inhibittranslation. Since ribozymes unlike antisense molecules, are catalytic,a lower intracellular concentration is required for efficiency. [0422]Endogenous gene expression can also be reduced by inactivating or“knocking out” the TR21 or TR22 gene and/or its promoter using targetedhomologous recombination. (E.g., see Smithies et al., Nature 317:230-234(1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart. The contents of each of the documents recited in this paragraph isherein incorporated by reference in its entirety. 10423] The techniquesof gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”) may beemployed to modulate the activities of TR21 or TR22 thereby effectivelygenerating agonists and antagonists of TR21 or TR22. See generally, U.S.Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458,and Patten et aL, Curr. Opinion Biotechnol. 8:724- 33 (1997); Harayama,Trends Biotechnol. 16(2):76-82 (1998); Hansson et al., J Mol. Biol.287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13(1998) (each of these patents and publications are hereby incorporatedby reference). In one embodiment, alteration of TR21 or TR22polynucleotides and corresponding polypeptides may be achieved by DNAshuffling. DNA shuffling involves the assembly of two or more DNAsegments into a desired TR21 or TR22 molecule by homologous, orsite-specific, recombination. In another embodiment, TR21 or TR22polynucleotides and corresponding polypeptides may be alterred by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of TR21 or TR22 may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules. In preferred embodiments, the heterologousmolecules are include, but are not limited to, TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), TRAIL, AIM-II (International Publication No. WO 97/34911),APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha (InternationalPublication No. WO 98/07880), neutrakine alpha (InternationalPublication No.WO98/18921), TR6 (International Publication No. WO98/30694), OPG, OX40, and nerve growth factor (NGF), and soluble formsof Fas, CD30, CD27, CD40 and 4-EBB, TR2 (International Publication No.WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4(International Publication No. WO 98/32856), TR5 (InternationalPublication No. WO 98/30693), TR6 (International Publication No. WO98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9(International Publication No. WO 98/56892), 312C2 (InternationalPublication No. WO 98/06842), and TR12, and soluble forms CD154, CD70,and CD153. In further preferred embodiments, the heterologous moleculesare any member of the TNF family. [0424] In other embodiments,antagonists according to the present invention include soluble forms ofTR21 or TR22 (e.g., fragments of the TR21 or TR22 shown in FIGS. 1A-Band 2, respectively, or encoded by the cDNA of deposited plasimdsHCFMV39 or HMUCLO1, respectively) that include the ligand binding domainfrom the extracellular region of the full length receptor). Such solubleforms of the TR21 or TR22, which may be naturally occurring orsynthetic, antagonize TR21 or TR22 mediated signaling by competing withthe cell surface bound forms of the receptor for binding to TNF-familyligands. Antagonists of the present invention also include antibodiesspecific for TNF- family ligands and TR21 or TR22-Fc fusion proteins.10425] By a “TNF-family ligand” is intended naturally occurring,recombinant, and synthetic ligands that are capable of binding to amember of the TNF receptor family and inducing and/or blocking theligand/receptor signaling pathway. Members of the TNF ligand familyinclude, but are not limited to, TNF-alpha, lymnphotoxin-alpha(LT-alpha, also known as TNF-beta), LT-beta (found in complexheterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL,DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328),TRAIL, AIM-l1 (International Publication No. WO 97/34911), APRIL (J.Exp. Med. 188(6):1185-1190), endokine-alpha (International PublicationNo. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG,OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30,CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095),DR3 (International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892), 312C2 (International Publication No. WO98/06842), and TR12, and soluble forms CD154, CD70, and CD153. [0426]TNF-aX has been shown to protect mice from infection with herpes simplexvirus type 1 (HSV-1). Rossol-Voth et al., J .Gen. Virol. 72:143-147(1991). The mechanism of the protective effect of TNF-a is unknown butappears to involve neither interferons nor NK cell killing. One memberof the family has been shown to mediate HSV-1 entry into cells.Montgomery et al., Eur. Cytokine Newt. 7:159 (1996). Further, antibodiesspecific for the extracellular domain of this block HSV-1 entry intocells. Thus, TR21 and TR22 antagonists of the present invention includeboth TR21 and TR22 amino acid sequences and antibodies capable ofpreventing mediated viral entry into cells. Such sequences andantibodies can finction by either competing with cell surface localizedfor binding to virus or by directly blocking binding of virus to cellsurface receptors. [04271 Antibodies according to the present inventionmay be prepared by any of a variety of methods using TR21 or TR22immunogens of the present invention. As indicated, such TR21 and TR22immunogens include the full length TR21 and TR22 polypeptide (which mayor may not include the leader sequence) and TR21 and TR22 polypeptidefragments such as the extracellular domain, the cysteine rich domain,the ligand binding domain, the transmembrane domain, the intracellulardomain and the incomplete death domain, or any combination thereof[04281 Polyclonal and monoclonal antibody agonists or antagonistsaccording to the present invention can be raised according to themethods disclosed herein and and/or known in the art, such as, forexample, those methods described in Tartaglia and Goeddel, J Biol. Chem.267(7):4304-4307(1992); Tartaglia et al., Cell 73:213-216 (1993), andPCT Application WO 94/09137 (the contents of each of these threepublications are herein incorporated by reference in their entireties),and are preferably specific to TR21 or TR22 polypeptides of theinvention having the amino acid sequence of Figures lA-B or 2,resepctively. [04291 An agonists according to the present inventioninclude soluble forms of TR21 or TR22, i.e., TR21 or TR22 fragments thatinclude the ligand binding domain from the extracellular region of thefull length receptor. Such soluble forms of the receptor, which may benaturally occurring or synthetic, antagonize TR2 1 or TR22 mediatedsignaling by competing with the cell surface TR21 or TR22 for binding toTNF-family ligands. However, soluble TR21 or TR22 may bind to apoptosisinducing ligands such as TRAIL and more effectively compete for TRAILbinding reducing the available TRAIL for binding to receptors withfunctional death domains. Thus, soluble forms of the receptor thatinclude the ligand binding domain are novel cytokines capable ofinhibiting apoptosis induced by TNF-family ligands. These are preferablyexpressed as dimers or trimers, since these have been shown to besuperior to monomeric forms of soluble receptor as antagonists, e.g.,IgGFc-TNF receptor family fusions. Other such cytokines are known in theart and include Fas B (a soluble form of the mouse Fas receptor) thatacts physiologically to limit apoptosis induced by Fas ligand (D.P.Hughes and I.N. Crispe, J Exp. Med. 182:1395-1401(1995)). [0430]Proteins and other compounds which bind the TR21 or TR22 extracellulardomains are also candidate agonists and antagonists according to thepresent invention. Such binding compounds can be “captured” using theyeast two-hybrid system (Fields and Song, Nature 340:245-246 (1989)). Amodified version of the yeast two- hybrid system has been described byRoger Brent and his colleagues (J. Gyuris, Cell 75:791-803 (1993); A.S.Zervos et al., Cell 72:223-232 (1993)). Preferably, the yeast two-hybridsystem is used according to the present invention to capture compoundswhich bind to either the TR21 or TR22 ligand binding domain or to theTR21 or TR22 intracellular domain. Such compounds are good candidateagonists and antagonists of the present invention. [0431] In rejectionof an allograft, the immune system of the recipient animal has notpreviously been primed to respond because the immune system for the mostpart is only primed by environmental antigens. Tissues from othermembers of the same species have not been presented in the same waythat, for example, viruses and bacteria have been presented. In the caseof allograft rejection, immunosuppressive regimens are designed toprevent the immune system from reaching the effector stage. However, theimmune profile of xenograft rejection may resemble disease recurrencemore that allograft rejection. In the case of disease recurrence, theimmune system has already been activated, as evidenced by destruction ofthe native islet cells. Therefore, in disease recurrence, the immunesystem is already at the effector stage. Antagonists of the presentinvention are able to suppress the immune response to both allograftsand xenografts because lymphocytes activated and differentiated intoeffector cells will express the TR21 orTR22 polypeptide, and thereby aresusceptible to compounds which enhance apoptosis. Thus, the presentinvention further provides a method for creating immune privilegedtissues. [0432] TR21 or TR22 antagonists of the invention can further beused in the treatment of inflammatory diseases, such as inflammatorybowel disease, rheumatoid arthritis, osteoarthritis, psoriasis, andsepticemia. In addition, due to lymphoblast expression of TR21 or TR22,soluble TR21 or TR22 agonist or antagonist mABs may be used to treatthis form of cancer. Modes ofAdministration [0433] The agonist orantagonists described herein can be administered in vitro, ex vivo, orin vivo to cells which express the receptor of the present invention. Byadministration of an “effective amount” of an agonist or antagonist isintended an amount of the compound that is sufficient to enhance orinhibit a cellular response to a TNF- family ligand and includepolypeptides. In particular, by administration of an “effective amount”of an agonist or antagonists is intended an amount effective to enhanceor inhibit TR21 or TR22 mediated apoptosis. Of course, where it isdesired for apoptosis to be enhanced, an agonist according to thepresent invention can be co-administered with a TNF-family ligand. Oneof ordinary skill will appreciate that effective amounts of an agonistor antagonist can be determined empirically and may be employed in pureform or in pharmaceutically acceptable salt, ester or prodrug form. Theagonist or antagonist may be administered in compositions in combinationwith one or more pharmaceutically acceptable excipients. [0434] It willbe understood that, when administered to a human patient, the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective dose level forany particular patient will depend upon factors well known in themedical arts. [0435] As a general proposition, the totalpharmaceutically effective amount of TR2 1 or TR22 polypeptideadministered parenterally per dose will be in the range of about 1ug/kg/day to 10 mg/kg/day of patient body weight, although, as notedabove, this will be subject to therapeutic discretion. More preferably,this dose is at least 0.01 mg/kg/day, and most preferably for humansbetween about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the TR21 or TR22 polypeptide is typically administered ata dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4injections per day or by continuous subcutaneous infusions, for example,using a mini-pump. An intravenous bag solution may also be employed.[04361 Dosaging may also be arranged in a patient specific manner toprovide a predetermined concentration of an agonist or antagonist in theblood, as determined by the RIA technique. Thus patient dosaging may beadjusted to achieve regular on-going trough blood levels, as measured byRIA, on the order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.[0437] Pharmaceutical compositions containing the TR21 or TR22polypeptide of the invention may be administered orally, rectally,parenterally, intracistemally, intravaginally, intraperitoneally,topically (as by powders, ointments, drops or transdermal patch),bucally, or as an oral or nasal spray. By “pharmaceutically acceptablecarrier” is meant a non-toxic solid, semisolid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrastemal,subcutaneous and intraarticular injection and infusion. [0438]Pharmaceutical compositions of the present invention for parenteralinjection can comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. [0439] In addition to soluble TR21 or TR22polypeptides, TR21 or TR22 polypeptides containing the transmembraneregion can also be used when appropriately solubilized by includingdetergents, such as CHAPS or NP-40, with buffer. [04401 TR21 or TR22compositions of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).10441] Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556(1983)), poly (2- hydroxyethyl methacrylate) (R. Langer et al., JBiomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D- (-)-3-hydroxybutyric acid (EP 133,988). [0442] Sustained-releasecompositions also include liposomally entrapped compositions of theinvention (see generally, Langer, Science 249:1527-1533 (1990); Treat etal., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 317 -327 and353-365 (1989)). Liposomes containing TR21 AND TR22 polypeptide my beprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046;EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes areof the small (about 200-800 Angstroms) unilamellar type in which thelipid content is greater than about 30 mol. percent cholesterol, theselected proportion being adjusted for the optimal TR21 or TR22polypeptide therapy. [0443] In yet an additional embodiment, thecompositions of the invention are delivered by way of a pump (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). [04441 Other controlled release systems are discussedin the review by Langer (Science 249:1527-1533 (1990)). [0445] Thecompositions of the invention may be administered alone or incombination with other adjuvants. Adjuvants that may be administeredwith the compositions of the invention include, but are not limited to,alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, compositionsof the invention are admninistered in combination with alum. In anotherspecific embodiment, compositions of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe compositions of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-18, CRL1005,Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines thatmay be administered with the compositions of the invention include, butare not limited to, vaccines directed toward protection against MMR(measles, mumps, rubella), polio, varicella, tetanus/diptheria,hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough,pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever,Japanese encephalitis, poliomyelitis, rabies, typhoid fever, andpertussis. Combinations may be administered either concomitantly, e.g.,as an admixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second. [0446] The compositions of the invention may beadministered alone or in combination with other therapeutic agents.Therapeutic agents that may be administered in combination with thecompositions of the invention, include but are not limited to, othermembers of the TNF family, chemotherapeutic agents, antibiotics,antivirals, steroidal and non-steroidal anti-inflammatories,conventional immunotherapeutic agents, cytokines, chemokines and/orgrowth factors. Combinations may be administered either concomitantly,e.g., as an admixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second. [0447] In one embodiment, the compositions ofthe invention are administered in combination with other members of theTNF family. TNF, TNF-related or TNF-like molecules that may beadministered with the compositions of the invention include, but are notlimited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha,also known as TNF-beta), LT-beta (found in complex heterotrimerLT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L,TNF-gamma (International Publication No. WO 96/14328), TRAIL, AIM-il(International Publication No. WO 97/34911), APRIL (J Exp. Med.188(6):1185-1190), endokine-alpha (International Publication No. WO98/07880), TR6 (International Publication No. WO 98/30694), OPG, OX40,and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27,CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892), 312C2 (International Publication No. WO98/06842), and TR12, and soluble forms CD154, CD70, and CD153. 10448] Incertain embodiments, compositions of the invention are administered incombination with antiretroviral agents, nucleoside reverse transcriptaseinhibitors, non- nucleoside reverse transcriptase inhibitors, and/orprotease inhibitors. Nucleoside reverse transcriptase inhibitors thatmay be administered in combination with the compositions of theinvention, include, but are not limited to, RETROVIRTM (zidovudine/AZT),VIDEXTM (didanosine/ddl), HIVIDTM (zalcitabine/ddC), ZERITTM(stavudine/d4T), EPIVIRTM (lamivudine/3TC), and COMBIVIRTM(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, V TM (nevirapine),RESCRIPTORTM (delavirdine), and SUSTIVATM (efavirenz). Proteaseinhibitors that may be administered in combination with the compositionsof the invention, include, but are not limited to, CRIXIVANTM(indinavir), NORVIRTM (ritonavir), INVIASETM (saquinavir), andVIRACEPTTM (nelfinavir). In a specific embodiment, antiretroviralagents, nucleoside reverse transcriptase inhibitors, non-nucleosidereverse transcriptase inhibitors, and/or protease inhibitors may be usedin any combination with compositions of the invention to treat AIDSand/or to prevent or treat HIV infection. 104491 In other embodiments,compositions of the invention may be administered in combination withanti-opportunistic infection agents. Anti-opportunistic agents that maybe administered in combination with the compositions of the invention,include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLETM,DAPSONETM, PENTAMDINETM, ATOVAQUONETM, ISONIAZIDTM, RHAMPIN M,PYRAZINAMIDETM, ETHAMBUTOL TM, RIFABUTIWM, CLARITHROMYCwm,AZITHROMYCINM, GANCICLOVIRTM, FOSCARNETTM, CIDOFOVIRTM, FLUCONAZOLETM,ITRACONAZOLETM, KETOCONAZOLETM, ACYCLOVIRTM, FAMCICOLVIRM,PYRIMETHAMINETM, LEUCOVORINTM, NEUPOGENM (filgrastim/G-CSF), andLEUKINETM (sargramostim/GM-CSF). In a specific embodiment, compositionsof the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINETM, and/or ATOVAQUONETM toprophylactically treat or prevent an opportunistic Pneumocystis cariniipneumonia infection. In another specific embodiment, compositions of theinvention are used in any combination with ISONIAZID TM, RIFAMPNTM,PYRAZINAMIDETM, and/or ETHAMBUTOLTM to prophylactically treat or preventan opportunistic Mycobacterium avium complex infection. In anotherspecific embodiment, compositions of the invention are used in anycombination with RIFABUTNOM, CLARITHROMYCINO, and/or AZITHROMYCIWM toprophylactically treat or prevent an opportunistic Mycobacteriumtuberculosis infection. In another specific embodiment, compositions ofthe invention are used in any combination with GANCICLOVIRTM,FOSCARNETTM, and/or CIDOFOVIRTM to prophylactically treat or prevent anopportunistic cytomegalovirus infection. In another specific embodiment,compositions of the invention are used in any combination withFLUCONAZOLET, ITRACONAZOLE, and/or KETOCONAZOLETM to prophylacticallytreat or prevent an opportunistic fungal infection. In another specificembodiment, compositions of the invention are used in any combinationwith ACYCLOVIRTM and/or FAMCICOLVIRTM to prophylactically treat orprevent an opportunistic herpes simplex virus type I and/or type IIinfection. In another specific embodiment, compositions of the inventionare used in any combination with PYRIMETHAMINETM and/or LEUCOVORINM toprophylactically treat or prevent an opportunistic Toxoplasma gondiiinfection. In another specific embodiment, compositions of the inventionare used in any combination with LEUCOVORINm and/or NEUPOGENTM toprophylactically treat or prevent an opportunistic bacterial infection.104501 In a further embodiment, the compositions of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the compositions of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine. [04511 In a further embodiment, the compositions of theinvention are administered in combination with an antibiotic agent.Antibiotic agents that may be administered with the compositions of theinvention include, but are not limited to, amoxicillin, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 1 5-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

Additional immunosuppressants preparations that may be administered withthe compositions of the invention include, but are not limited to,ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclosporin), PROGRAF™(tacrolimus), CELLCEPT™ (mycophenolate), Azathioprine,glucorticosteroids, and RAPAMUNE™ (sirolimus). In a specific embodiment,immunosuppressants may be used to prevent rejection of organ or bonemarrow transplantation.

In an additional embodiment, compositions of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the compositions of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, compositions of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the compositionsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compostions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, compositions of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, compositions of the invention are administered incombination with Rituximab. In a further embodiment, compositions of theinvention are administered with Rituxmab and CHOP, or Rituxmab and anycombination of the components of CHOP.

In an additional embodiment, the compositions of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, GM-CSF, G-CSF, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-19, IL-20, IL-21, anti-CD40, CD40L, IFN-gamma andTNF-alpha. In one embodiment, the compositions of the invention areadministered in combination with one or more chemokines. In specificembodiments, the compositions of the invention are administered incombination with an α(CxC) chemokine selected from the group consistingof gamma-interferon inducible protein-10 (γIP-10), interleukin-8 (IL-8),platelet factor-4 (PF4), neutrophil activating protein (NAP-2), GRO-α,GRO-β, GRO-γ, neutrophil-activating peptide (ENA-78), granulocytechemoattractant protein-2 (GCP-2), and stromal cell-derived factor-1(SDF-1, or pre-B cell stimulatory factor (PBSF)); and/or a O(CC)chemokine selected from the group consisting of: RANTES (regulated onactivation, normal T expressed and secreted), macrophage inflammatoryprotein-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta(MIP-1β), monocyte chemotactic protein-1 (MCP-1), monocyte chemotacticprotein-2 (MCP-2), monocyte chemotactic protein-3 (MCP-3), monocytechemotactic protein-4 (MCP-4) macrophage inflammatory protein-1 gamma(MIP-1γ), macrophage inflammatory protein-3 alpha (MIP-3α), macrophageinflammatory protein-3 beta (MIP-3β), macrophage inflammatory protein-4(MIP-4/DC-CK-1/PARC), eotaxin, Exodus, and I-309; and/or the γ(C)chemokine, lymphotactin.

In a preferred embodiment, the compositions of the invention areadministered in combination with Stem Cell Factor or IL-3. In a mostpreferred embodiment the compositions of the invention are administeredin combination with Stem Cell Factor and IL-3.

In an additional embodiment, the compositions of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the compositions of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

The invention also encompasses combining the polynucleotides and/orpolypeptides of the invention (and/or agonists or antagonists thereof)with other proposed or conventional hematopoietic therapies. Thus, forexample, the polynucleotides and/or polypeptides of the invention(and/or agonists or antagonists thereof) can be combined with compoundsthat singly exhibit erythropoietic stimulatory effects, such aserythropoietin, testosterone, progenitor cell stimulators, insulin-likegrowth factor, prostaglandins, serotonin, cyclic AMP, prolactin, andtriiodothyzonine. Also encompassed are combinations of the compositionsof the invention with compounds generally used to treat aplastic anemia,such as, for example, methenolene, stanozolol, and nandrolone; to treatiron-deficiency anemia, such as, for example, iron preparations; totreat malignant anemia, such as, for example, vitamin B₁₂ and/or folicacid; and to treat hemolytic anemia, such as, for example,adrenocortical steroids, e.g., corticoids. See e.g., Resegotti et al.,Panminerva Medica, 23:243-248 (1981); Kurtz, FEBS Letters, 14a:105-108(1982); McGonigle et al., Kidney Int., 25:437-444 (1984); andPavlovic-Kantera, Expt. Hematol., 8(supp. 8) 283-291 (1980), thecontents of each of which are hereby incorporated by reference in theirentireties.

Compounds that enhance the effects of or synergize with erythropoietinare also useful as adjuvants herein, and include but are not limited to,adrenergic agonists, thyroid hormones, androgens, hepatic erythropoieticfactors, erythrotropins, and erythrogenins, See for e.g., Dunn, “CurrentConcepts in Erythropoiesis”, John Wiley and Sons (Chichester, England,1983); Kalmani, Kidney Int., 22:383-391 (1982); Shahidi, New Eng. J.Med., 289:72-80 (1973); Urabe et al., J. Exp. Med., 149:1314-1325(1979); Billat et al., Expt. Hematol., 10:133-140 (1982); Naughton etal., Acta Haemat, 69:171-179 (1983); Cognote et al. in abstract 364,Proceedings 7th Intl. Cong. of Endocrinology (Quebec City, Quebec, Jul.1-7, 1984); and Rothman et al., 1982, J. Surg. Oncol., 20:105-108(1982). Methods for stimulating hematopoiesis comprise administering ahematopoietically effective amount (i.e., an amount which effects theformation of blood cells) of a pharmaceutical composition containingpolynucleotides and/or poylpeptides of the invention (and/or agonists orantagonists thereof) to a patient. The polynucleotides and/orpolypeptides of the invention and/or agonists or antagonists thereof isadministered to the patient by any suitable technique, including but notlimited to, parenteral, sublingual, topical, intrapulmonary andintranasal, and those techniques further discussed herein. Thepharmaceutical composition optionally contains one or more members ofthe group consisting of erythropoietin, testosterone, progenitor cellstimulators, insulin-like growth factor, prostaglandins, serotonin,cyclic AMP, prolactin, triiodothyzonine, methenolene, stanozolol, andnandrolone, iron preparations, vitamin B₁₂, folic acid and/oradrenocortical steroids.

In additional prefered embodiments, the compositions of the inventionare administered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with thecompositions of the invention included, but are not limited to, LEUKINE™(SARGRAMOSTIM™) and NEUPOGEN™ (FELGRASTIM™).

In additional embodiments, the compositions of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Chromosome Assays

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. The mapping of DNAs to chromosomes according to the presentinvention is an important first step in correlating those sequences withgenes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a TR21 or TR22 receptor gene.This can be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAis then used for in situ chromosome mapping using well known techniquesfor this purpose.

In addition, in some cases, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes.

Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bp. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man, available on line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1

Expression and Purirfcation of the TR21 and TR22 Receptors in E. coli

The bacterial expression vector pQE60 is used for bacterial expressionin this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA,91311). pQE60 encodes ampicillin antibiotic resistance (“Amp^(r)”) andcontains a bacterial origin of replication (“ori”), an IPTG induciblepromoter, a ribosome binding site (“RBS”), six codons encoding histidineresidues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that a DNA fragment encoding apolypeptide may be inserted in such as way as to produce thatpolypeptide with the six His residues (i.e., a “6 X His tag”) covalentlylinked to the carboxyl terminus of that polypeptide. However, in thisexample, the polypeptide coding sequence is inserted such thattranslation of the six His codons is prevented and, therefore, thepolypeptide is produced with no 6 X His tag.

The DNA sequence encoding the desired portion of the TR21 or TR22protein lacking the hydrophobic leader sequence is amplified from thedeposited CDNA clone using PCR oligonucleotide primers which anneal tothe amino terminal sequences of the desired portion of the TR21 or TR22protein and to sequences in the deposited construct 3′ to the cDNAcoding sequence. Additional nucleotides containing restriction sites tofacilitate cloning in the pQE60 vector are added to the 5′ and 3′sequences, respectively.

For cloning the mature protein, the 5′ primer has the sequence:5′-CGCCCATGGGACAATGGGAGCAGCAGC-3′ (SEQ ID NO: 8)containing the underlined NcoI restriction site followed by nucleotidescomplementary to the amino terminal coding sequence of the mature TR21sequence in FIGS. 1A-B. One of ordinary skill in the art wouldappreciate, of course, that the point in the protein coding sequencewhere the 5′ primer begins may be varied to amplify a desired portion ofthe complete protein shorter or longer than the mature form.

The 3′ primer has the sequence: (SEQ ID NO: 9)5′-CGCAAGCTTCACCAAAACCACGGCTCACCTG-3′containing the underlined Hindill site followed by nucleotidescomplementary to the 3′ end of the non-coding sequence in the TR21 DNAsequence in FIG. 1A-B.

The amplified TR21 or TR22 DNA fragments and the vector pQE60 aredigested with Nco I and HindIII and the digested DNAs then ligatedtogether. Insertion of the TR21 or TR22 protein DNA into the restrictedpQE60 vector places the TR21 or TR22 protein coding region (includingits associated stop codon) downstream from the IPTG-inducible promoterand in-frame with an initiating AUG. The associated stop codon preventstranslation of the six histidine codons downstream of the insertionpoint.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures. Such procedures are described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kan^(T)”), isused in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressing TR21or TR22 protein, is available commercially from Qiagen, Inc., supra.

Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR, and DNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 ug/ml)and kanamycin (25 ug/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:100 to 1:250. The cells aregrown to an optical density at 600nm (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacl repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

The cells are then stirred for 3-4 hours at 4° C in 6M guanidine-HCI,pH8. The cell debris is removed by centrifugation, and the supernatantcontaining the TR21 or TR22 is loaded onto a nickel-nitrilo-tri-aceticacid (“NiNTA”) affinity resin column (available from QIAGEN, Inc.,supra). Proteins with a 6×His tag bind to the NI-NTA resin with highaffinity and can be purified in a simple one-step procedure (for detailssee: The QIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly thesupernatant is loaded onto the column in 6 M guanidine-HCl, pH8, thecolumn is first washed with 10 volumes of 6 M guanidine-HCl, pH8, thenwashed with 10 volumes of 6 M guanidine-HCl pH6, and finally the TR21 orTR22 is eluted with 6 M guanidine-HCl, pH5.

The purified protein is then renatured by dialyzing it againstphosphatebuffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200mM NaCl. Alternatively, the protein can be successfully refolded whileimmobilized on the Ni-NTA column. The recommended conditions are asfollows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20%glycerol, 20 mM Tris/HCl pH7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins can be eluted by the addition of 250 mMimmidazole. Inmidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

Example 2

Cloning and Expression of TR21 or TR22 in a Baculovirus ExpressionSystem

In this illustrative example, the plasmid shuttle vector pA2 is used toinsert the cloned DNA encoding the complete protein, including itsnaturally associated secretary signal (leader) sequence, into abaculovirus to express the mature TR21 or TR22 protein, using standardmethods as described in Summers et al., A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No. 1555 (1987). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI and Asp7l8. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate viable virus thatexpress the cloned polynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcINM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

The cDNA sequence encoding the mature TR21 receptor protein, forexample, in the deposited clone HCFMV39, lacking the AUG initiationcodon and the naturally associated leader sequence shown in FigureslA-B, is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene.

The 5′ primer has a sequence containing the underlined BamHI restrictionenzyme site, an efficient signal for initiation of translation ineukaryotic cells, as described by M. Kozak, J. Mol. Biol. 196:947- 950(1987), followed by bases of the sequence of the mature TR21 proteinshown in FIGS. 1A-B, beginning with the indicated N-terminus of themature protein.

The 3′ primer for has a sequence containing the underlined Asp7l8restriction site followed by nucleotides complementary to the 3′noncoding sequence in FIGS. 1A-B.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean, ” BIO 101 Inc., La Jolla,Calif.) The fragment then is digested with BamHI and Asp718 and again ispurified on a 1% agarose gel. This fragment is designated “F1.”

The plasmid is digested with the restriction enzyme Barn HI andoptionally can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.). The vector DNA is designated herein “V1.”

Fragment F1 and the dephosphorylated plasmid V1 are ligated togetherwith T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts suchas XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria are identified that contain the plasmid with the human TR21 orTR22 gene using the PCR method, in which one of the primers that is usedto amplify the gene and the second primer is from well within the vectorso that only those bacterial colonies containing the TR21 or TR22 genefragment will show amplification of the DNA. The sequence of the clonedfragment is confirmed by DNA sequencing. This plasmid is designatedherein pBacTR21 or TR22.

Five ug of the plasmid pBacTR21 or TR22 is co-transfected with 1.0 ug ofa commercially available linearized baculovirus DNA (“BaculoGoldTmbaculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectinmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417 (1987). 1 ug of BaculoGold™ virus DNA and 5 ug of theplasmid pBacTR21 or TR22 are mixed in a sterile well of a microliterplate containing 50 ul of serum free Grace's medium (Life Technologies,Inc., Rockville, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace'smedium are added, mixed, and incubated for 15 minutes at roomtemperature. Then, the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours, the transfection solution is removed from theplate and 1 ml of Grace's insect medium supplemented with 10% fetal calfserum is added. The plate is put back into an incubator and cultivationis continued at 27° C. for four days.

After four days, the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, cited above. An agarosegel with “Blue Gal” (Life Technologies, Inc., Rockville, Md.) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies, Inc.,Rockville, Md., pages 9-10). After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant virus is called V-TR21 or TR22.

To verify the expression of the TR21 or TR22 gene, Sf9 cells are grownin Grace's medium supplemented with 10% heat inactivated FBS. The cellsare infected with the recombinant baculovirus V-TR21 or TR22 at amultiplicity of infection (“MOI”) of about 2. Six hours later the mediumis removed and is replaced with SF900 II medium minus methionine andcysteine (available from Life Technologies, Inc., Rockville, Md.). Ifradiolabeled proteins are desired, 42 hours later, 5 uCi of³⁵S-methionine and 5 uCi 35S cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled). Microsequencing of the amino acidsequence of the amino terminus of purified protein may be used todetermine the amino terminal sequence of the mature protein and thus thecleavage point and length of the secretory signal peptide.

Example 3

Cloning and Expression of the TR21 and TR22 Receptors in Mammalian Cells

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g. RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular signals can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells, andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. Co-transfection with aselectable marker such as dhfr, gpt, neomycin, or hygromycin allows theidentification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The dihydrofolate reductase (DHFR) marker is usefulto develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy et al., Biochem. J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance selected. These celllines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) cells are often used for the production ofproteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology5:438-447 (March 1985)), plus a fragment of the CMV-enhancer (Boshart etal., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

Example 3A

Cloning and Expression of the Extracellular Soluble Domain of TR21 andTR22 in COS cells

The expression plasmid, pTR21-HA or pTR22-HA, is made by cloning a cDNAencoding TR21 or TR22 into the expression vector pcDNAI/Amp or pcDNAIH(which can be obtained from Invitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcell; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron, and a polyadenylation signal arranged so that a cDNAconveniently can be placed under expression control of the CMV promoterand operably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker.

A DNA fragment encoding the entire TR21 or TR22 precursor and a HA tagfused in frame to its 3′ end is cloned into the polylinker region of thevector so that recombinant protein expression is directed by the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein described by Wilson et al., Cell 37:767(1984). The fusion of the HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is as follows:

The TR21 or TR22 cDNA of the deposited clone is amplified using primersthat contain convenient restriction sites, much as described aboveregarding the construction of expression vectors for expression of TR21and TR22 in E. coli.

To facilitate detection, purification and characterization of theexpressed TR21 or TR22, one of the primers contains a hemagglutinin tag(“HA tag”) as described above.

Suitable primers for TR21 and TR22 include the following, which are usedin this example:

The 5′ primer contains the a BamHI site, followed by an ATG start codonand 5 codons for TR21 or TR22 thereafter. The 3′ primer for TR21 or TR22contains an XbaI site, stop codon, hemagglutinin tag, and the last 19nucleotides of the 3′ coding sequence (at the 3′ end).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith BamHI and XbaI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037) thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisand gel sizing for the presence of the TR21 or TR22-encoding fragment.

For expression of recombinant TR21 or TR22, COS cells are transfectedwith an expression vector, as described above, using DEAE-DEXTRAN, asdescribed, for instance, in Sambrook et al., Molecular Cloning: aLaboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor,N.Y. (1989). Cells are incubated under conditions for expression of TR21or TR22 by the vector.

Expression of the TR21 or TR22-HA fusion protein is detected byradiolabelling and immunoprecipitation, using methods described in, forexample Harlow et al., Antibodies: a Laboratory Manual, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and then lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE gels and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3B

Cloning and Expression of TR21 and TR22 using the CHO Expression System

The vector pC4 is used for the expression of the TR21 or TR22polypeptide. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCCAccession No. 37146). The plasmid contains the mouse DHFR gene undercontrol of the SV40 early promoter. Chinese hamster ovary- or othercells lacking dihydrofolate activity that are transfected with theseplasmids can be selected by growing the cells in a selective medium(alpha minus MEM, Life Technologies, Rockville, Md.) supplemented withthe chemotherapeutic agent methotrexate (MTX). The amplification of theDHFR genes in cells resistant to MTX has been well documented (see,e.g., F. W. Alt et al., J. Biol. Chem. 253:1357-1370 (1978); J. L.Hamlin and C. Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); M. J.Page M. A. Sydenham, Biotechnology 9:64-68(1991)). Cells grown inincreasing concentrations of MTX develop resistance to the drug byoverproducing the target enzyme, DHFR, as a result of amplification ofthe DHFR gene. If a second gene is linked to the DHFR gene, it isusually co-amplified and over-expressed. It is known in the art thatthis approach may be used to develop cell lines carrying more than 1,000copies of the amplified gene(s). Subsequently, when the methotrexate iswithdrawn, cell lines are obtained that contain the amplified geneintegrated into one or more chromosome(s) of the host cell.

Plasmid pC4 contains, for expressing the gene of interest, the strongpromoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus(Cullen et al., Molecular and Cellular Biology 5:438-447 (March 1985)),plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530 (1985)).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, XbaI, andAsp718. Behind these cloning sites, the plasmid contains the 3′ intronand the polyadenylation site of the rat preproinsulin gene. Other highefficiency promoters can also be used for the expression, e.g., thehuman B-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the TR21 or TR22 polypeptide in aregulated way in mammalian cells. For the polyadenylation of the mRNA,other signals, e.g., from the human growth hormone or globin genes, canbe used as well.

Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418, or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzyme BamHI and thendephosphorylated using calf intestinal phosphates, by procedures knownin the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete TR21 or TR22 polypeptide isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ sequences of the desired portion of the gene.

The 5′ oligonucleotide primer for TR21 or TR22, contains a BamHIrestriction site, a Kozak sequence, and an AUG start codon. The 3′primer for TR21 or TR22, contains an Asp718 restriction site followed bythe reverse complement of the 3′ untranslated region of the gene.

The amplified fragment is digested with BamHI and then purified again ona 1% agarose gel. The isolated fragment and the dephosphorylated vectorare then ligated with T4 DNA ligase. E. coli HB11101 or XL-1 Blue cellsare then transformed and bacteria are identified that contain thefragment inserted into plasmid pC4 using, for instance, restrictionenzyme analysis.

Chinese hamster ovary cells lacking an active DHFR enzyme are used fortransfection. Five ug of the expression plasmid pC4 are cotransfectedwith 0.5 ug of the plasmid pSVneo using the lipofectin method (Felgneret al., supra). The plasmid pSV2-neo contains a dominant selectablemarker, the neo gene from Tn5 encoding an enzyme that confers resistanceto a group of antibiotics including G418. The cells are seeded in alphaminus MEM supplemented with 1 mg/ml G418. After 2 days, the cells aretrypsinized and seeded in hybridoma cloning plates (Greiner, Germany) inalpha minus MEM supplemented with 10, 25, or 50 ng/ml of MTX plus 1mg/ml G418. After about 10-14 days, single clones are trypsinized andthen seeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 uM, 2 uM, 5 uM, 10 uM, 20 uM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 uM. Expression of the desired gene product is analyzed, forinstance, by Western blot analysis and SDS-PAGE, or by reversed phaseHPLC analysis.

Example 4

Protein Fusions of TR21 and TR22

TR21 and TR22 polypeptides of the invention are optionally fused toother proteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of TR21 or TR22 polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See EP A 394,827; Traunecker, et al., Nature331:84-86 (1988)). Similarly, fusion to IgG-1, IgG-3, and albuminincreases the halflife time in vivo. Nuclear localization signals fusedto TR21 or TR22 polypeptides can target the protein to a specificsubcellular localization, while covalent heterodimer or homodimers canincrease or decrease the activity of a fusion protein. Fusion proteinscan also create chimeric molecules having more than one function.Finally, fusion proteins can increase solubility and/or stability of thefused protein compared to the non-fused protein. All of the types offusion proteins described above can be made using techniques known inthe art or by using or routinely modifying the following protocol, whichoutlines the fusion of a polypeptide to an IgG molecule. Furthermore,human serum albumin, which can be fused to the polypeptides andfragments of the invention, is described in U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentireties. [05191 Briefly, the human Fc portion of the IgG molecule canbe PCR amplified, using primers that span the 5′ and 3′ ends of thesequence described below (SEQ ID NO:16). These primers also preferablycontain convenient restriction enzyme sites that will facilitate cloninginto an expression vector, preferably a mammalian expression vector.

For example, if the pC4 (Accession No. 209646) expression vector isused, the human Fc portion can be ligated into the BainHI cloning site.Note that the 3′ BamHI site should be destroyed. Next, the vectorcontaining the human Fc portion is re-restricted with BamHI, linearizingthe vector, and TR21 or TR22 polynucleotide, isolated by the PCRprotocol described in Example 1, is ligated into this BamHI site. Notethat the polynucleotide is cloned without a stop codon, otherwise afusion protein will not be produced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Human IgG Fc region: (SEQ ID NO: 10)        GGGATCCGGAGCCGAAATGTTCTGACAAAACTCACACATGCCCACCGTGCCCAGGACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCAGGAAGACCGTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGAGCCGGAGAACAACTACAAGACGACGCCTCCCGTGGTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAGCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 5

TR21 and TR22 Induced Apoptosis

Experimental Design

To facilitate detection, TR21 or TR22 is cloned into pCMV2FLAG (1131Kodak) as an in-frame fusion to the signal sequence and FLAG-epitope tagencoded by the vector. The cDNA encoding the extracellular domain ofTR21 or is obtained by PCR, similar to the methods described above, andsubcloned into a modified pCMVlFLAG vector that allowed for in-framefusion with the Fc portion of human IgG. DR4-Fc, TNRF1-Fc, Fc andsoluble TRAIL and TNF alpha expression constructs have been describedpreviously, Pan, G. et al., Science 276:111-113 (1997), which isincorporated herein by reference in its entirety.

The receptor-Fc fusions and soluble ligands are prepared and in vivobinding is performed as previously described, Pan G. et al., Science276:111-113 (1997), and Pan G. et al., Science 277:815-818 (1997), bothof which are incorporated herein by reference in their entirety.

Cell death blocking assays using receptor-Fc fusions are carried out asdescribed previously by Pan G. et al., Science 276:111-113 (1997), andPan G. et al., Science 277:815-818 (1997), both of which areincorporated herein by reference in their entirety.

The extracellular domain of TR21 or TR22 is expressed as a secretedchimera fused to the Fc portion of human IgG in 293 cells. Conditionedmedium from transfected cells is mixed with bacterially expressedsoluble His-FLAG-tagged TRAIL. The resulting complex is precipitatedwith protein G-Sepharose and bound TRAIL detected by Western blottingwith anti-FLAG antibody.

Example 6

Production of an Antibody

Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing TR21 or TR22 are administered to an animal toinduce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of TR21 or TR22 protein is prepared andpurified to render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

Monoclonal antibodies specific for TR21 or TR22 protein are preparedusing hybridoma technology. (Kohler et al., Nature 256:495 (1975);Kohler et al., Eur. J. bnmunol. 6:511(1976); Kohler et al., Eur. J.Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies andT-Cell Hybridomas, Elsevier, N.Y., pp. 563-681(1981)). In general, ananimal (preferably a mouse) is immunized with TR21 or TR22 polypeptideor, more preferably, with a secreted TR21 or TR22 polypeptide-expressingcell. Such polypeptide-expressing cells are cultured in any suitabletissue culture medium, preferably in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 g/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP20), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981). The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the TR21 or TR22polypeptide.

Alternatively, additional antibodies capable of binding to TR21 or TR22polypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the TR21 or TR22protein-specific antibody can be blocked by TR21 or TR22. Suchantibodies comprise anti-idiotypic antibodies to the TR21 or TR22protein-specific antibody and are used to immunize an animal to induceformation of further TR21 or TR22 protein-specific antibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric and humanized antibodies are known in theart and are discussed infra. (See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Patent No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).)

Isolation of Antibody Fragments Directed Against TR21 and TR22 from aLibrary of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstTR21 or TR22 to which the donor may or may not have been exposed (seee.g., U.S. Pat. No. 5,885,793 incorporated herein by reference in itsentirety).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 109 E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100 jig/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 withshaking. Five ml of this culture is used to innoculate 50 ml of2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene HI, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 pg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene HI protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type genemll protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (EEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2×TY broth containing 100 gg ampicillin/ml and 25,g kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 [tg/ml or 10 tig/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing.

Example 7

Tissue Distribution of TR21 and TR22 mRNA Expression

Northern blot analysis is carried out to examine TR21 and TR22 geneexpression in human tissues, using methods described by, among others,Sambrook et al., cited above. A cDNA probe containing the entirenucleotide sequence of the TR21 or TR22 polypeptide coding sequence islabeled with ³²p using the rediprimeTM DNA labeling system (AmershamLife Science), according to manufacturer's instructions. After labeling,the probe is purified using a CHROMA SPIN-100 column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe is then used to examine varioushuman tissues for TR21 or TR22 mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) are obtained from Clontech andare examined with labeled probe using ExpressHyb™ hybridization solution(Clontech) according to manufacturer's protocol number PT1190-1.Following hybridization and washing, the blots are mounted and exposedto film at −70° C. overnight, and films developed according to standardprocedures.

Northern Blot Analysis of TR21 and TR22 in Various Cell Lines Methods:Cells

HL60 (promyelocytic leukemia), Hela cell S3, K562 (chronic myelogeneousleukemia), MOLT4 (lymphoblast leukemia), Raji (Burkitt's lyrnphoma),SW480 (colorectal adenocarcinoma), A549 (lung carcinoma), and G361(melanoma), cell lines are obtained from the American Type CultureCollection (Rockville, Md.). Primary carotid artery endothelial cellsare purchased from Clonetics Corp. (San Diego, Calif.) and monocytes areprepared by differential centrifugation of peripheral blood mononuclearcells and adhesion to tissue culture dish. CD19+, CD4+ and CD8+ cells(>90% pure) are isolated with cell type specific immunomagnetic beads(Drynal, Lake Success, NY).

RNA Analysis

Total RNA of adult tissues are purchased from Clonetech (Palo Alto,Calif.). Total RNA is extracted from cell lines (in exponential growthphase) and primary cells with TiiReagent (Molecular Research Center,Inc., Cinciimati, Ohio). 5 to 7.5 ug of total RNA is fractionated in a1% agarose gel containing formaldehyde cast in a Wide Mini-Sub Cell geltray (Bio-Rad, Hercules, Calif.) as described (Sambrook, et al.) withslight modifications. The formaldehyde concentration is reduced to 0.5Mand the RNA is stained prior to electrophoresis with 100 ug/ml ofethidium bromide that is added to the loading buffer. Afterelectrophoresis with continuous buffer recirculation (60 volts/90 min),the gel is photographed and the RNA is transferred quantitatively toZeta-probe nylon membrane (Biorad, Hercules, Calif.) by vacuum-blottingwith 25 mM NaOH for 90 min. After neutralization for 5-10 min, with I1MTris-HCl, pH 7.5 containing 3M NaCl, the blots are prehybridized with50% formnamide, 8% dextran sulfate, 6xSSPE, 0.1% SDS and 100 ug/ml ofsheared and denatured salmon sperm DNA for at least 30 min at 42° C.cDNA inserts labeled with 32 P-DCTP by random priming (Stratagene, LaJolla, Calif.), are denatured with 0.25M NaOH (IO min at 37° C.) andadded to the prehybridization solution. After 24-65 hr at 42° C., theblots are washed under high stringency conditions (Sambrook, et al.) andexposed to X-ray films.

Example 8

Method of Determining Alterations in the TR21 and TR22 Genes

RNA is isolated from entire families or individual patients presentingwith a phenotype of interest (such as a disease). cDNA is then generatedfrom these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in Figures IA-B or 2. SuggestedPCR conditions consist of 35 cycles at 95° C. for 30 seconds; 60-120seconds at 52-58° C.; and 60-120 seconds at 70° C., using buffersolutions described in Sidransky, D., et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons ofTR21 or TR22 are also determined and genomic PCR products analyzed toconfirm the results. PCR products harboring suspected mutations in TR21or TR22 is then cloned and sequenced to validate the results of thedirect sequencing.

PCR products of TR21 or TR22 are cloned into T-tailed vectors asdescribed in Holton, T. A. and Graham, M. W., Nucleic Acids Research,19:1156 (1991) and sequenced with T7 polymerase (United StatesBiochemical). Affected individuals are identified by mutations in TR21or TR22 not present in unaffected individuals. Genomic rearrangementsare also observed as a method of determining alterations in the TR21 orTR22 gene. Genomic clones isolated using techniques known in the art arenick-translated with digoxigenindeoxy-uridine 5′-triphosphate(Boehringer Manheim), and FISH performed as described in Johnson, Cg. etal., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeledprobe is carried out using a vast excess of human cot-I DNA for specifichybridization to the TR21 or TR22 genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, AZ) and variableexcitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, NC.) Chromosomealterations of the genomic region of TR21 or TR22 (hybridized by theprobe) are identified as insertions, deletions, and translocations.These TR21 or TR22 alterations are used as a diagnostic marker for anassociated disease.

Example 9

Method ofDetecting Abnormal Levels of TR21 and TR22 in a BiologicalSample

TR21 and TR22 polypeptides can be detected in a biological sample, andif an increased or decreased level of TR21 or TR22 is detected, thispolypeptide is a marker for a particular phenotype. Methods of detectionare numerous, and thus, it is understood that one skilled in the art canmodify the following assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect TR21 or TR22 ina sample, preferably a biological sample. Wells of a microtiter plateare coated with specific antibodies to TR21 or TR22, at a finalconcentration of 0.2 to 10 ug/ml. The antibodies are either monoclonalor polyclonal and are produced using technique known in the art. Thewells are blocked so that non-specific binding of TR21 or TR22 to thewell is reduced. [05531 The coated wells are then incubated for >2 hoursat RT with a sample containing TR21 or TR22. Preferably, serialdilutions of the sample should be used to validate results. The platesare then washed three times with deionized or distilled water to removeunbounded TR21 or TR22.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate(NPP) substrate solution is then added to each well and incubated 1 hourat room temperature to allow cleavage of the substrate and flourescence.The flourescence is measured by a microtiter plate reader. A standardcurve is preparded using the experimental results from serial dilutionsof a control sample with the sample concentration plotted on the X-axis(log scale) and fluorescence or absorbance on the Y-axis (linear scale).The TR21 or TR22 polypeptide concentration in a sample is theninterpolated using the standard curve based on the measured flourescenceof that sample.

Example 10

Method of Treating Decreased Levels of TR21 and TR22

The present invention relates to a method for treating an individual inneed of a decreased level of TR21 or TR22 biological activity in thebody comprising, administering to such an individual a compositioncomprising a therapeutically effective amount of TR21 or TR22antagonist. Preferred antagonists for use in the present invention areTR21 or TR22-specific antibodies.

Moreover, it will be appreciated that conditions caused by a decrease inthe standard or normal expression level of TR21 or TR22 in an individualcan be treated by administering TR21 or TR22, preferably in a solubleand/or secreted form. Thus, the invention also provides a method oftreatment of an individual in need of an increased level of TR21 or TR22polypeptide comprising administering to such an individual apharmaceutical composition comprising an amount of TR21 or TR22 toincrease the biological activity level of TR21 or TR22 in such anindividual.

For example, a patient with decreased levels of TR21 or TR22 polypeptidereceives a daily dose 0.1-100 ug/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in a soluble and/orsecreted form.

Example 11

Method of Treating Increased Levels of TR21 and TR22

The present invention also relates to a method for treating anindividual in need of an increased level of TR21 and TR22 biologicalactivity in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of TR21 andTR22 or an agonist thereof.

Antisense technology is used to inhibit production of TR21 or TR22. Thistechnology is one example of a method of decreasing levels of TR21 orTR22 polypeptide, preferably a soluble and/or secreted form, due to avariety of etiologies, such as cancer.

For example, a patient diagnosed with abnormally increased levels ofTR21 or TR22 is administered intravenously antisense polynucleotides at0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment isrepeated after a 7-day rest period if the is determined to be welltolerated.

Example 12

Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing soluble and/or mature TR21 or TR22 polypeptides, onto apatient. Generally, fibroblasts are obtained from a subject by skinbiopsy. The resulting tissue is placed in tissue-culture medium andseparated into small pieces. Small chunks of the tissue are placed on awet surface of a tissue culture flask, approximately ten pieces areplaced in each flask. The flask is turned upside down, closed tight andleft at room temperature over night. After 24 hours at room temperature,the flask is inverted and the chunks of tissue remain fixed to thebottom of the flask and fresh media (e.g., Ham's F12 media, with 10%FBS, penicillin and streptomycin) is added. The flasks are thenincubated at 37 C for approximately one week.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and Hindifi and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding TR21 or TR22 can be amplified using PCR primers whichcorrespond to the 5′ and 3′ end encoding sequences respectively.Preferably, the 5′ primer contains an EcoRI site and the 3′ primerincludes a HindIII site. Equal quantities of the Moloney murine sarcomavirus linear backbone and the amplified EcoRI and Hindli fragment areadded together, in the presence of T4 DNA ligase. The resulting mixtureis maintained under conditions appropriate for ligation of the twofragments. The ligation mixture is then used to transform E. coli HB101,which are then plated onto agar containing kanamycin for the purpose ofconfirming that the vector contains properly inserted TR21 or TR22.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the TR21 or TR22 gene is then added to the media and thepackaging cells transduced with the vector. The packaging cells nowproduce infectious viral particles containing the TR21 or TR22 gene (thepackaging cells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether TR21 or TR22protein is produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 13

Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) TR21 orr TR22 sequences into an animal to increaseor decrease the expression of the TR21 or TR22 polypeptide. The TR21 orTR22 polynucleotide may be operatively linked to a promoter or any othergenetic elements necessary for the expression of the TR21 or TR22polypeptide by the target tissue. Such gene therapy and deliverytechniques and methods are known in the art, see, for example,W090/11092, W098/11779; U.S. Patent Nos. 5693622, 5705151, 5580859;Tabata H. et al., Cardiovasc. Res. 35:470-479 (1997); Chao J. et al.,Pharmacol. Res. 35:517-522 (1997); Wolff J. A. Neuromuscul. Disord.7:314-318 (1997); Schwartz B. et al., Gene Ther. 3:405-411(1996);Tsurumi Y. et al., Circulation 94:3281-3290 (1996) (incorporated hereinby reference).

The TR21 or TR22 polynucleotide constructs may be delivered by anymethod that delivers injectable materials to the cells of an animal,such as, injection into the interstitial space of tissues (heart,muscle, skin, lung, liver, intestine and the like). The TR21 or TR22polynucleotide constructs can be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the TR21 or TR22 polynucleotides may also bedelivered in liposome formulations (such as those taught in FelgnerP.L., et al. Ann. NY Acad. Sci. 772:126-139 (1995), and Abdallah B., etal. Biol. Cell 85(1):1-7 (1995)) which can be prepared by methods wellknown to those skilled in the art.

The TR21 or TR22 polynucleotide vector constructs used in the genetherapy method are preferably constructs that will not integrate intothe host genome nor will they contain sequences that allow forreplication. Any strong promoter known to those skilled in the art canbe used for driving the expression of DNA. Unlike other gene therapiestechniques, one major advantage of introducing naked nucleic acidsequences into target cells is the transitory nature of thepolynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The TR21 or TR22 polynucleotide construct can be delivered to theinterstitial space of tissues within the an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non- dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

For the naked TR21 or TR22 polynucleotide injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 g/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked TR21 or TR22polynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected TR21 or TR22 polynucleotide inmuscle in vivo is determined as follows. Suitable TR21 or TR22 templateDNA for production of mRNA coding for TR21 or TR22 polypeptide isprepared in accordance with a standard recombinant DNA methodology. Thetemplate DNA, which may be either circular or linear, is either used asnaked DNA or complexed with liposomes. The quadriceps muscles of miceare then injected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The TR21 or TR22 template DNA is injected in 0.1 ml ofcarrier in a I cc syringe through a 27 gauge needle over one minute,approximately 0.5 cm from the distal insertion site of the muscle intothe knee and about 0.2 cm deep. A suture is placed over the injectionsite for future localization, and the skin is closed with stainlesssteel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for TR21 or TR22 protein expression. A time course for TR21 orTR22 protein expression may be done in a similar fashion except thatquadriceps from different mice are harvested at different times.Persistence of TR21 or TR22 DNA in muscle following injection may bedetermined by Southern blot analysis after preparing total cellular DNAand HIRT supernatants from injected and control mice. The results of theabove experimentation in mice can be use to extrapolate proper dosagesand other treatment parameters in humans and other animals using TR21 orTR22 naked DNA.

Example 14

Gene Therapy Using Endogenous TR21 or TR22 Gene

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous TR21 or TR22 sequence witha promoter via homologous recombination as described, for example, inU.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International PublicationNumber WO 96/29411, published Sep. 26, 1996; International PublicationNumber WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl.Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature342:435-438 (1989). This method involves the activation of a gene whichis present in the target cells, but which is not expressed in the cells,or is expressed at a lower level than desired. Polynucleotide constructsare made which contain a promoter and targeting sequences, which arehomologous to the 5′ non-coding sequence of endogenous TR21 or TR22,flanking the promoter. The targeting sequence will be sufficiently nearthe 5′ end of TR21 or TR22 so the promoter will be operably linked tothe endogenous sequence upon homologous recombination. The promoter andthe targeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenousTR21 or TR22 sequence. This results in the expression of TR21 or TR22 inthe cell. Expression may be detected by immunological staining, or anyother method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the TR21 or TR22 locus, plasmidpUC18 (MBI Fermentas, Amherst, N.Y.) is digested with Hindli. The CMVpromoter is amplified by PCR with an XbaI site on the 5′ end and a BamHIsite on the 3′end. Two TR21 or TR22 non-coding sequences are amplifiedvia PCR: one TR21 or TR22 non-coding sequence (TR21 or TR22 fragment 1)is amplified with a Hindifi site at the 5′ end and an Xba site at the3′end; the other TR21 or TR22 non-coding sequence (TR21 or TR22 fragment2) is amplified with a BamHI site at the 5′end and a Hindlil site at the3′end. The CMV promoter and TR21 or TR22 fragments are digested with theappropriate enzymes (CMV promoter—XbaI and BamHI; TR2 or TR22 fragment1—XbaI; TR21 or TR22 fragment 2—BamHI) and ligated together. Theresulting ligation product is digested with HindIll, and ligated withthe HindEi-digested pUC 18 plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 gg/ml.0.5 ml of the cell suspension (containing approximately 1.5×10⁶cells) isthen added to the cuvette, and the cell suspension and DNA solutions aregently mixed. Electroporation is performed with a Gene-Pulser apparatus(Bio-Rad). Capacitance and voltage are set at 960 JF and 250-300 V,respectively. As voltage increases, cell survival decreases, but thepercentage of surviving cells that stably incorporate the introduced DNAinto their genome increases dramatically. Given these parameters, apulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 C. The following day, the media is aspiratedand replaced with 10 ml of fresh media and incubated for a further 16-24hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 15

Bioassay for the Effect of TR21 and TR22 Polypeptides, Agonists, orAntagonists on Hematopoietic Progenitor Cells and/or Differentiation.

Mouse bone marrow cells are used as target cells to examine the effectof TR21 or TR22 polypeptides of the invention on hematopoieticprogenitor cells and/or differentiation. Briefly, unfractionated bonemarrow cells are first washed 2× with a serum-free IMDM that issupplemented with 10% (V/V) BIT (Bovine serum albumin, Insulin andTransferrin supplement from Stem Cell Technologies, Vancouver, Canada).The washed cells are then resuspended in the same growth medium andplated in the 96-well tissue culture plate (5×10⁴ cells/well) in 0.2 mlof the above medium in the presence or absence of cytokines and TR21 orTR22. Stem cell factor (SCF) and IL-3 are included as positive mediatorsof cell proliferation. Cells are allowed to grow in a low oxygenenvironment (5% CO₂, 7% O², and 88% N₂) tissue culture incubator for 6days. On the sixth day, 0.5 pCi of Tritiated thymidine is added to eachwell and incubation is continued for an additional 16-18 hours, at whichpoint the cells are harvested. The level of radioactivity incorporatedinto cellular DNA is determined by scintillation spectrometry andreflects the amount of cell proliferation.

The studies described in this example test the activity of TR21 and TR22polypeptides of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of TR21 andTR22 polynucleotides (e.g., gene therapy), agonists, and/or antagonistsof TR21 and TR22. Potential agonists would be expected to inhibithematopoietic cell proliferation in the presence of SCF and/or IL3and/or to increase the inhibition of cell proliferation in the presenceof cytokines and TR21 or TR22 in this assay. Potential antagonists wouldbe expected to reduce the inhibition of cell proliferation in thepresence of cytokines and TR21 or TR22 in this assay.

Example 16

Effect of TR21 and TR22-fc Fusion (Le., Chimera) Polypeptides onHematopoietic Progenitor Cells and/or Differentiation

Mouse bone marrow cells are used as target cells to examine the effectof TR21 and TR22 polypeptides of the invention on hematopoieticprogenitor cells and/or differentiation. Briefly, unfractionated bonemarrow cells are first washed 2× with a serum-free IMDM that issupplemented with 10% (V/V) BIT (Bovine serum albumin, Insulin andTransferrin supplement from Stem Cell Technologies, Vancouver, Canada).The washed cells are then resuspended in the same growth medium, platedin a 96-well tissue culture plate (5×10⁴ cells/well) in 0.2 ml of theabove medium in the presence or absence of cytokines and TR21 or TR22.Stem cell factor (SCF) and IL-3 are included as positive mediators ofcell proliferation. Cells are allowed to grow in a low oxygenenvironment (5% CO₂, 7% O², and 88% N₂) tissue culture incubator for 6days. On the sixth day, 0.5 pCi of Tritiated thymidine is added to eachwell and incubation is continued for an additional 16-18 hours, at whichpoint the cells are harvested. The level of radioactivity incorporatedinto cellular DNA is determined by scintillation spectrometry andreflects the amount of cell proliferation.

Example 17

Bioassay for the Effect of TR21 and TR22 Polypeptides, Agonists orAntagonists on IL-3 and SCF Stimulated Proliferation and Differentiationof Hematopoietic Progenitor Cells.

To determine if TR21 or TR22 polypeptides of the invention inhibitspecific hematopoietic lineages, mouse bone marrow cells are firstwashed 2× with a serum-free IMDM that is supplemented with 10% (V/V) BIT(Bovine serum albumin, Insulin and Transferrin supplement from Stem CellTechnologies, Vancouver, Canada). The washed cells are then resuspendedin the same growth medium and plated in the 96-well tissue culture plate(5×10⁴ cells/well) in 0.2 ml of the above medium in the presence of IL-3(1 ng/ml) plus SCF (5 ng/ml) with or without TR2 1 or TR22. Cells areallowed to grow in a low oxygen environment (5% CO₂, 7% O², and 88% N₂)tissue culture incubator, and after 7 days, analyzed for expression ofdifferentiation antigens by staining with various monoclonal antibodiesand FACScan.

The studies described in this example test the activity of TR21 and TR22polypeptides of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of TR21 andTR22 polynucleotides (e.g., gene therapy), agonists, and/or antagonistsof TR21 and TR22. Potential agonists tested in this assay would beexpected to inhibit cell proliferation in the presence of cytokinesand/or to increase the inhibition of cell proliferation in the presenceof cytokines and TR21 or TR22. Potential antagonists tested in thisassay would be expected to reduce the inhibition of cell proliferationin the presence of cytokines and TR21 or TR22.

Example 18

Effect of TR21 and TR22-Fc Chimera on IL-3 and SCF StimulatedProliferation and Differentiation of Hematopoietic Progenitor Cells.

To determine if TR21 or TR22 polypeptides of the invention inhibitspecific hematopoietic lineages, mouse bone marrow cells are firstwashed 2X with a serum-free IMDM that is supplemented with 10% (VN) BIT(Bovine serum albumin, Insulin and Transferrin supplement from Stem CellTechnologies, Vancouver, Canada). The washed cells are then resuspendedin the same growth medium and plated in 96-well tissue culture plate(5×10⁴ cells/well) in 0.2 ml of the above medium in the presence of IL-3(1 ng/ml) plus SCF (5 ng/ml) with or without TR21 or TR22-Fc. Cells areallowed to grow in a low oxygen environment (5% CO₂, 7% O², and 88% N₂)tissue culture incubator, and after 7 days, analyzed for expression ofdifferentiation antigens by staining with various monoclonal antibodiesand FACScan.

Example 19

Effect of TR21 AND TR22 on IL-3 and SCF Stimulated Proliferation andDifferentiation of Lin-Population of Bone Marrow Cells

A population of mouse bone marrow cells enriched in primitivehematopoietic progenitors can be obtained using a negative selectionprocedure, where the committed cells of most of the lineages are removedusing a panel of monoclonal antibodies (anti cd11b, CD4, CD8, CD45R andGr-1 antigens) and magnetic beads. The resulting population of cells(lineage depleted cells) are plated (5×10⁴ cells/ml) in the presence orabsence of TR21 or TR22 polypeptide of the invention (in a range ofconcentrations) in a growth medium supplemented with IL-3 (5 ng/ml) plusSCF (100 ng/ml). After seven days of incubation at 37 C in a humidifiedincubator (5% CO₂, 7% O₂, and 88% N₂ environment), cells are harvestedand assayed for the HPP-CFC, and immature progenitors. In addition,cells are analyzed for the expression of certain differentiationantigens by FACScan. Colony data is expressed as mean number of colonies±SD) and are obtained from assays performed in six dishes for eachpopulation of cells.

Example 20

Assays to Detect Stimulation or Inhibition of B Cell Proliferation andDifferentiation

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL5, IL6,IL-7, ILIO, IL-13, IL14 and IL15. Interestingly, these signals are bythemselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection andlor observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

In Vitro assay—Purified TR21 or TR22 polylpeptides of the invention(e.g., soluble TR21 AND TR22) or agonists or antagonists thereof, areassessed for their ability to induce activation, proliferation,differentiation or inhibition and/or death in B-cell populations andtheir precursors. The activity of TR21 or TR22 polypeptides, or agonistsor antagonists thereof on purified human tonsillar B cells, measuredqualitatively over the dose range from 0.1 to 10,000 ng/ml, is assessedin a standard B-lymphocyte co- stimulation assay in which purifiedtonsillar B cells are cultured in the presence of either formalin-fixedStaphylococcus aureus Cowan I (SAC) or immobilized anti-human IgMantibody as the priming agent. Second signals such as IL-2 and IL-15synergize with SAC and IgM crosslinking to elicit B cell proliferationas measured by tritiated-thymidine incorporation. Novel synergizingagents can be readily identified using this assay. The assay involvesisolating human tonsillar B cells by magnetic bead (MACS) depletion ofCD3-positive cells. The resulting cell population is greater than 95% Bcells as assessed by expression of CD45R(B220). Various dilutions ofeach sample are placed into individual wells of a 96-well plate to whichare added 105 B-cells suspended in culture medium (RPMI 1640 containing10% FBS, 5×10⁻⁵M βME, 100 U/ml penicillin, 10 ug/ml streptomycin, and10⁻⁵ dilution of SAC) in a total volume of 150 ul. Proliferation orinhibition is quantitated by a 20 h pulse (1 uCi/well) with ³H-thymidine(6.7 Ci/mM) beginning 72h post factor addition. The positive andnegative controls are IL2 and medium respectively.

In Vivo assay- BALB/c mice are injected (i.p.) twice per day with bufferonly, or 2 mg/Kg of TR21 or TR22 polypeptide (e.g., soluble TR21 orTR22) or agonists or antagonists thereof. Mice receive this treatmentfor 4 consecutive days, at which time they are sacrificed and varioustissues and serum collected for analyses. Comparison of H&E sectionsfrom normal and TR21 or TR22 polypeptide-treated spleens identify theresults of the activity of TR21 or TR22 polypeptide on spleen cells,such as the diffusion of peri- arterial lymphatic sheaths, and/orsignificant increases in the nucleated cellularity of the red pulpregions, which may indicate the activation of the differentiation andproliferation of B-cell populations. Immunohistochemical studies using aB cell marker, anti-CD45R(B220), are used to determine whether anyphysiological changes to splenic cells, such as splenic disorganization,are due to increased B-cell representation within loosely defined B-cellzones that infiltrate established T-cell regions.

Flow cytometric analyses of the spleens from TR21 or TR22polypeptide-treated mice is used to indicate whether TR21 or TR22polypeptide specifically increases the proportion of ThB+,CD45R(B220)dull B cells over that which is observed in control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andTR21 or TR22 polypeptide-treated mice.

The studies described in this example test the activity in TR21 and TR22polypeptides. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), and agonists, and/or antagonistsof TR21 and TR22.

Example 21

T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of ³H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100 el/well of nAb to CD3 (HIT3a,Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4° C. (1jig/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three timeswith PBS. PBMC are isolated by F/H gradient centrifugation from humanperipheral blood and added to quadruplicate wells (5×10⁴/well) of mAbcoated plates in RPMI containing 10% FCS and P/S in the presence ofvarying concentrations of TR21 or TR22 protein (total volume 200 μl).Relevant protein buffer and medium alone are controls. After 48 hr.culture at 37° C., plates are spun for 2 min. at 1000 rpm and 100 μl ofsupernatant is removed and stored −20° C. for measurement of IL-2 (orother cytokines) if effect on proliferation is observed. Wells aresupplemented with 100 μl of medium containing 0.5 ,Ci of ³H-thymidineand cultured at 37° C. for 18-24 hr. Wells are harvested andincorporation of ³H-thymidine used as a measure of proliferation.Anti-CD3 alone is the positive control for proliferation. IL-2 (100U/ml) is also used as a control which enhances proliferation. Controlantibody which does not induce proliferation of T cells is used as thenegative controls for the effects of TR21 or TR22 proteins.

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 22

Effect of TR21 and TR22 on the Expression of MHC Class II, Costimulatoryand Adhesion Molecules and Cell Differentiation of monocytes andMonocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-α,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFCγRII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1-3 days with increasing concentrations of TR21 or TR22 or LPS(positive control), washed with PBS containing 1% BSA and 0.02 mM sodiumazide, and then incubated with 1:20 dilution of appropriate FITC- orPE-labeled monoclonal antibodies for 30 minutes at 4° C. After anadditional wash, the labeled cells are analyzed by flow cytometry on aFACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendriticcells, in particular IL-12, are important in the initiation of T-celldependent immune responses. IL-12 strongly influences the development ofThl helper T-cell immune response, and induces cytotoxic T and NK cellfunction. An ELISA is used to measure the IL-12 release as follows.Dendritic cells (10⁶/ml) are treated with increasing concentrations ofTR21 or TR22 for 24 hours. LPS (100 ng/ml) is added to the cell cultureas positive control. Supernatants from the cell cultures are thencollected and analyzed for IL-12 content using commercial ELISA kit(e.g., R & D Systems (Minneapolis, MN)). The standard protocols providedwith the kits are used.

Effect on the expression of MHC Class II, costimulatory and adhesionmolecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations of TR21 orTR22 or LPS (positive control), washed with PBS containing 1% BSA and0.02 mM sodium azide, and then incubated with 1:20 dilution ofappropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at4° C. After an additional wash, the labeled cells are analyzed by flowcytometry on a FACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules thatactivate (or alternatively, inactivate) monocytes and/or increasemonocyte survival (or alternatively, decrease monocyte survival) areknown in the art and may routinely be applied to determine whether amolecule of the invention functions as an inhibitor or activator ofmonocytes. TR21 and TR22, agonists, or antagonists of TR21 and TR22 canbe screened using the three assays described below. For each of theseassays, Peripheral blood mononuclear cells (PBMC) are purified fromsingle donor leukopacks (American Red Cross, Baltimore, Md.) bycentrifugation through a Histopaque gradient (Sigma). Monocytes areisolated from PBMC by counterflow centrifugal elutriation.

1. Monocyte Survival Assay. Human peripheral blood monocytesprogressively lose viability when cultured in absence of serum or otherstimuli. Their death results from internally regulated process(apoptosis). Addition to the culture of activating factors, such asTNF-alpha dramatically improves cell survival and prevents DNAfragmentation. Propidium iodide (PI) staining is used to measureapoptosis as follows. Monocytes are cultured for 48 hours inpolypropylene tubes in serum-free medium (positive control), in thepresence of 100 ng/ml TNF-alpha (negative control), and in the presenceof varying concentrations of the compound to be tested. Cells aresuspended at a concentration of 2 x 10⁶/ml in PBS containing PI at afinal concentration of 5 μg/ml, and then incubated at room temperaturefor 5 minutes before FAC Scan analysis. PI uptake has been demonstratedto correlate with DNA fragmentation in this experimental paradigm.

2. Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5x10⁵ cells/mlwith increasing concentrations of TR21 or TR22 and under the sameconditions, but in the absence of TR21 or TR22. For IL-12 production,the cells are primed overnight with IFN-γ (100 U/ml) in presence of TR21or TR22. LPS (10 ng/ml) is then added. Conditioned media are collectedafter 24h and kept frozen until use. Measurement of TNF-a, IL-10, MCP-1and IL-8 is then performed using a commercially available ELISA kit(e.g., R & D Systems (Minneapolis, Minn.)) applying the standardprotocols provided with the kit.

3. Oxidative burst. Purified monocytes are plated in 96-well plate at2-1×10¹⁵ cell/well. Increasing concentrations of TR21 or TR22 are addedto the wells in a total volume of 0.2 ml culture medium (RPMI 1640+10%FCS, glutamine and antibiotics). After 3 days incubation, the plates arecentrifuged and the medium is removed from the wells. To the macrophagemonolayers, 0.2 ml per well of phenol red solution (140 mM NaCI, 10 mMpotassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol redand 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA).The plates are incubated at 37° C. for 2 hours and the reaction isstopped by adding 20 μl IN NaOH per well. The absorbance is read at 610nm. To calculate the amount of H₂O₂ produced by the macrophages, astandard curve of a H₂O₂ solution of known molarity is performed foreach experiment.

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 23

The Effect of TR21 and TR22 on the Growth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at2-5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. TR21 orTR22 protein, and positive controls, such as VEGF and basic FGF (bFGF)are added, at varying concentrations. On days 4 and 6, the medium isreplaced. On day 8, cell number is determined with a Coulter Counter. Anincrease in the number of HUVEC cells indicates that TR21 or TR22 mayproliferate vascular endothelial cells.

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 24

Stimulatory Effect of TR21 and TR22 on the Proliferation of VascularEndothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny1)2H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 ml serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF₁₆₅ or TR21 or TR22 in 0.5% FBS) with orwithout Heparin (8 U/ml) are added to wells for 48 hours. 20 mg ofMTS/PMS mixture (1:0.05) are added per well and allowed to incubate for1 hour at 37° C. before measuring the absorbance at 490 nm in an ELISAplate reader. Background absorbance from control wells (some media, nocells) is subtracted, and seven wells are performed in parallel for eachcondition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518(1994).

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 25

Inhibition of PDGF-induced Vascular Smooth Muscle Cell ProliferationStimulatory Effect

HAoSMC proliferation upon treatment with TR21 or TR22 can be measured,for example, by BrdUrd incorporation. Briefly, subconfluent, quiescentcells grown on the 4-chamber slides are transfected with CRP orFITC-labeled AT2-3LP. Then, the cells are pulsed with 10% calf serum and6 mg/ml BrdUrd. After 24 h, immunocytochemistry is performed by usingBrdUrd Staining Kit (Zymed Laboratories). In brief, the cells areincubated with the biotinylated mouse anti-BrdUrd antibody at 4° C. for2 h after exposing to denaturing solution and then with thestreptavidin-peroxidase and diaminobenzidine. After counterstaining withhematoxylin, the cells are mounted for microscopic examination, and theBrdUrd-positive cells are counted. The BrdUrd index is calculated as apercent of the BrdUrd-positive cells to the total cell number. Inaddition, the simultaneous detection of the BrdUrd staining (nucleus)and the FITC uptake (cytoplasm) is performed for individual cells by theconcomitant use of bright field illumination and dark field-UVfluorescent illumination. See, Hayashida et al., J. Biol. Chem.6;271(36):21985-21992 (1996).

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 26

Stimulation of Endothelial Migration

This example will be used to explore the possibility that TR21 or TR22may stimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, Md.; Falk, W.,Goodwin, R. H. J., and Leonard, E. J. “A 48 well micro chemotaxisassembly for rapid and accurate measurement of leukocyte migration.” J.Immunological Methods 1980;33:239-247). Polyvinylpyrrolidone-freepolycarbonate filters with a pore size of 8 um (Nucleopore Corp.Cambridge, Mass.) are coated with 0.1% gelatin for at least 6 hours atroom temperature and dried under sterile air. Test samples of TR21 orTR22 are diluted to appropriate concentrations in M199 supplemented with0.25% bovine serum albumin (BSA), and 25 ul of the final dilution isplaced in the lower chamber of the modified Boyden apparatus.Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed andtrypsinized for the minimum time required to achieve cell detachment.After placing the filter between lower and upper chamber, 2.5×10⁵ cellssuspended in 50 ul M199 containing 1% FBS are seeded in the uppercompartment. The apparatus is then incubated for 5 hours at 37° C. in ahumidified chamber with 5% CO2 to allow cell migration. After theincubation period, the filter is removed and the upper side of thefilter with the non-migrated cells is scraped with a rubber policeman.The filters are fixed with methanol and stained with a Giemsa solution(Diff-Quick, Baxter, McGraw Park, Ill.). Migration is quantified bycounting cells of three random high-power fields (40×) in each well, andall groups are performed in quadruplicate.

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 27

Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be amediator of vascular endothelium relaxation. Thus, TR21 and TR22activity can be assayed by determining nitric oxide production byendothelial cells in response to TR21 or TR22.

Nitric oxide is measured in 96-well plates of confluent microvascularendothelial cells after 24 hours starvation and a subsequent 4 hrexposure to various levels of a positive control (such as VEGF-1) andTR21 or TR22. Nitric oxide in the medium is determined by use of theGriess reagent to measure total nitrite after reduction of nitricoxide-derived nitrate by nitrate reductase. The effect of TR21 or TR22on nitric oxide release is examined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with aNO-specific polarographic electrode connected to a NO meter (Iso-NO,World Precision Instruments Inc.). Calibration of the NO element isperformed according to the following equation:2KNO₂+2KI+2H₂SO₄62NO+I₂+2H₂O+2K₂SO₄

The standard calibration curve is obtained by adding gradedconcentrations of KNO2 (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing KI and H₂SO₄. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas. The culture medium is removed and HVECs arewashed twice with Dulbecco's phosphate buffered saline. The cells arethen bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) to maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×10⁶ endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 28

and TR22 on Cord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications' CADMEC Growth Medium and used at passage 5. Forthe in vitro angiogenesis assay, the wells of a 48-well cell cultureplate are coated with Cell Applications Attachment Factor Medium (200μl/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 μg Cell Applications' Chord FormationMedium containing control buffer or TR21 or TR22 (0.1 to 100 ng/ml) andthe cells are cultured for an additional 48 hr. The numbers and lengthsof the capillary-like chords are quantitated through use of theBoeckeler VIA-1 70 video image analyzer. All assays are done intriplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.b-esteradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 29

Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system toexamine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of TR21 or TR22 to stimulate angiogenesisin CAM can be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and theJapanese quail (Coturnix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old quailembryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chickeggs. The embryos are checked for normal development and the eggs sealedwith cellotape. They are further incubated until Day 13. Thermanoxcoverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm indiameter. Sterile and salt-free growth factors and TR21 or TR22 aredissolved in distilled water and about 3.3 mg/ 5 ml are pipetted on thedisks. After air-drying, the inverted disks are applied on CAM. After 3days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehydeand rinsed in 0.12 M sodium cacodylate buffer. They are photographedwith a stereo microscope [Wild M8] and embedded for semi- and ultrathinsectioning as described above. Controls are performed with carrier disksalone.

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 30

Angiogenesis Assay Using a Matrigel Implant in Mouse

In order to establish an in vivo model for angiogenesis to test TR21 andTR22 protein activities, mice and rats are implanted subcutaneously withmethylcellulose disks containing either 20 mg of BSA (negative control),1 mg of TR21 or TR22, or 0.5 mg of VEGF-1 (positive control). Thenegative control disks should contain little vascularization, while thepositive control disks should show signs of vessel formation.

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 31

Rescue ofIschemia in Rabbit Lower Limb Model

To study the in vivo effects of TR21 and TR22 on ischemia, a rabbithindlimb ischemia model is created by surgical removal of one femoralarteries as described previously (Takeshita, S. et al., Am J. Pathol147:1649-1660 (1995)). The excision of the femoral artery results inretrograde propagation of thrombus and occlusion of the external iliacartery. Consequently, blood flow to the ischemic limb is dependent uponcollateral vessels originating from the internal iliac artery(Takeshita, S. et al., Am J. Pathol 147:1649-1660 (1995)). An intervalof 10 days is allowed for post-operative recovery of rabbits anddevelopment of endogenous collateral vessels. At 10 day post-operatively(day 0), after performing a baseline angiogram, the internal iliacartery of the ischemic limb is transfected with 500 mg naked TR21 orTR22 expression plasmid by arterial gene transfer technology using ahydrogel-coated balloon catheter as described (Riessen, R. et al., HumGene Ther. 4:749-758 (1993); Leclerc, G. et al., J Clin. Invest. 90:936-944 (1992)). When TR21 or TR22 is used in the treatment, a singlebolus of 500 mg TR21 or TR22 protein or control is delivered into theinternal iliac artery of the ischemic limb over a period of 1 min.through an infusion catheter. On day 30, various parameters are measuredin these rabbits: (a) BP ratio—The blood pressure ratio of systolicpressure of the ischemic limb to that of normal limb; (b) Blood Flow andFlow Reserve—Resting FL: the blood flow during undilated condition andMax FL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount) and Flow Reserve is reflected by theratio of max FL: resting FL; (c) Angiographic Score—This is measured bythe angiogram of collateral vessels. A score is determined by thepercentage of circles in an overlaying grid that with crossing opacifiedarteries divided by the total number m the rabbit thigh; (d) Capillarydensity—The number of collateral capillaries determined in lightmicroscopic sections taken from hindlimbs.

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 32

Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, andfactor VIII immunohistochemistry or endothelial alkaline phosphatasereaction. TR21 or TR22 expression, during the skin ischemia, is studiedusing in situ hybridization.

The study in this model is divided into three parts as follows:

-   -   Ischemic skin    -   Ischemic skin wounds    -   Normal wounds

The experimental protocol includes:

Raising a 3×4 cm, single pedicle full-thickness random skin flap(myocutaneous flap over the lower back of the animal).

An excisional wounding (4-6 mm in diameter) in the ischemic skin(skin-flap).

Topical treatment with TR21 or TR22 of the excisional wounds (day 0, 1,2, 3, 4 post-wounding) at the following various dosage ranges: lmg to100 mg.

Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21 post-woundingfor histological, immunohistochemical, and in situ studies.

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 33

Peripheral Arterial Disease Model

Angiogenic therapy using TR21 or TR22 is a novel therapeutic strategy toobtain restoration of blood flow around the ischemia in case ofperipheral arterial diseases. The experimental protocol includes:

One side of the femoral artery is ligated to create ischemic muscle ofthe hindlimb, the other side of hindlimb serves as a control.

TR21 or TR22 protein, in a dosage range of 20 mg-500 mg, is deliveredintravenously and/or intramuscularly 3 times (perhaps more) per week for2-3 weeks.

The ischemic muscle tissue is collected after ligation of the femoralartery at 1, 2, and 3 weeks for the analysis of TR21 or TR22 expressionand histology. Biopsy is also performed on the other side of normalmuscle of the contralateral hindlimb.

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 34

Ischemic Myocardial Disease Model

TR21 or TR22 is evaluated as a potent mitogen capable of stimulating thedevelopment of collateral vessels, and restructuring new vessels aftercoronary artery occlusion. Alteration of TR21 or TR22 expression isinvestigated in situ. The experimental protocol includes:

The heart is exposed through a left-side thoracotomy in the rat.Immediately, the left coronary artery is occluded with a thin suture(6-0) and the thorax is closed.

TR21 or TR22 protein, in a dosage range of 20 mg-500 mg, is deliveredintravenously and/or intramuscularly 3 times (perhaps more) per week for2-4 weeks.

Thirty days after the surgery, the heart is removed and cross-sectionedfor morphometric and in situ analyzes.

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 35

Rat Corneal Wound Healing Model

This animal model shows the effect of TR21 or TR22 onneovascularization. The experimental protocol includes:

Making a 1-1.5 mm long incision from the center of cornea into thestromal layer.

Inserting a spatula below the lip of the incision facing the outercorner of the eye.

Making a pocket (its base is 1-1.5 mm form the edge of the eye).

Positioning a pellet, containing 50ng-5ug of TR21 or TR22, within thepocket.

TR21 or TR22 treatment can also be applied topically to the comealwounds in a dosage range of 20mg-500mg (daily treatment for five days).

The studies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22.

Example 36

Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models

A. Diabetic db+/db+ Mouse Model.

To demonstrate that TR21 or TR22 accelerates the healing process, thegenetically diabetic mouse model of wound healing is used. The fullthickness wound healing model in the db+/db+ mouse is a wellcharacterized, clinically relevant and reproducible model of impairedwound healing. Healing of the diabetic wound is dependent on formationof granulation tissue and re-epithelialization rather than contraction(Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D.G. etal., Am. J Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L., Diabetes 31 (Suppl):1-6 -(1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andwere 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. mediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

TR21 or TR22 is administered using at a range different doses of TR21 orTR22, from 4mg to 500mg per wound per day for 8 days in vehicle. Vehiclecontrol groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and inununohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) TR21 or TR22.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 was 64 mm², the corresponding size of the dermalpunch. Calculations were made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (Smm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with TR21 or TR22. This assessment included verification ofthe presence of cell accumulation, inflammatory cells, capillaries,fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh,D.G. et al., Am. J Pathol. 136:1235 (1990)). A calibrated lensmicrometer is used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer served as a positive tissue controland human brain tissue is used as a negative tissue control. Eachspecimen included a section with omission of the primary antibody andsubstitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, S.M. Glucocorticoids andWound healing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahl, S. M. et al., J. Immunol. 115: 476-481(1975); Werb, Z. et al., J. Exp. Med. 147:1684-1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability ( Ebert, R. H., et al., An. Intern.Med. 37:701-705 (1952)), fibroblast proliferation, and collagensynthesis (Beck, L. S. et al., Growth Factors. 5: 295-304 (1991);Haynes, B. F. et al., J. Clin. Invest. 61: 703-797 (1978)) and producinga transient reduction of circulating monocytes (Haynes, B. F., et al.,J. Clin. Invest. 61: 703-797 (1978); Wahl, S. M., “Glucocorticoids andwound healing”, In: Antiinflammatory Steroid Action: Basic and ClinicalAspects, Academic Press, New York, pp. 280-302 (1989)). The systemicadministration of steroids to impaired wound healing is a well establishphenomenon in rats (Beck, L. S. et al., Growth Factors. 5: 295-304(1991); Haynes, B. F., et al., J Clin. Invest. 61: 703-797 (1978); Wahl,S. M., “Glucocorticoids and wound healing”, In: Antiinflammatory SteroidAction: Basic and Clinical Aspects, Academic Press, New York, pp.280-302 (1989); Pierce, G.F. et al., Proc. NatL. Acad. Sci. USA 86:2229-2233 (1989)). [0681] To demonstrate that TR21 or TR22 canaccelerate the healing process, the effects of multiple topicalapplications of TR21 or TR22 on full thickness excisional skin wounds inrats in which healing has been impaired by the systemic administrationof methylprednisolone is assessed. [06821 Young adult male SpragueDawley rats weighing 250-300 g (Charles River Laboratories) are used inthis example. The animals are purchased at 8 weeks of age and were 9weeks old at the beginning of the study. The healing response of rats isimpaired by the systemic administration of methylprednisolone (17mg/kg/rat intramuscularly) at the time of wounding. Animals areindividually housed and received food and water ad libitum. Allmanipulations are performed using aseptic techniques. This study isconducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals. 10683J Thewounding protocol is followed according to section A, above. On the dayof wounding, animals are anesthetized with an intramuscular injection ofketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of theanimal is shaved and the skin washed with 70% ethanol and iodinesolutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges. [06841 Wounds arevisually examined and photographed at a fixed distance at the day ofwounding and at the end of treatment. Wound closure is determined bydaily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson, caliper. Woundsare considered healed if granulation tissue was no longer visible andthe wound is covered by a continuous epithelium. [06851 TR21 or TR22 isadministered using at a range different doses of TR21 or TR22, from 4mgto 500mg per wound per day for 8 days in vehicle. Vehicle control groupsreceived 5OmL of vehicle solution. [0686] Animals are euthanized on day8 with an intraperitoneal injection of sodium pentobarbital (300mg/kg).The wounds and surrounding skin are then harvested for histology. Tissuespecimens are placed in 10% neutral buffered formalin in tissuecassettes between biopsy sponges for further processing. [06871 Fourgroups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) were evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) TR21 or TR22 treated groups. [06881 Wound closure is analyzedby measuring the area in the vertical and horizontal axis and obtainingthe total area of the wound. Closure is then estimated by establishingthe differences between the initial wound area (day 0) and that of posttreatment (day 8). The wound area on day 1 was 64mm2, the correspondingsize of the dermal punch. Calculations were made using the followingformula:

[Open area on day 8] - [Open area on day 1] / [Open area on day 1][0689] Specimens are fixed in 10% buffered formalin and paraffinembedded blocks are sectioned perpendicular to the wound surface (5mm)and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E)staining was performed on cross-sections of bisected wounds. Histologicexamination of the wounds allows assessment of whether the healingprocess and the morphologic appearance of the repaired skin was improvedby treatment with TR21 or TR22. A calibrated lens micrometer is used bya blinded observer to determine the distance of the wound gap. [0690]Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant. [06911 The studies described in thisexample test the activity in TR21 and TR22 protein. However, one skilledin the art could easily modify the exemplified studies to test theactivity of TR21 and TR22 polynucleotides (e.g., gene therapy),agonists, and/or antagonists of TR21 and TR22. Example 3 7 LymphademaAnimal Model [0692] The purpose of this experimental approach is tocreate an appropriate and consistent lymphedema model for testing thetherapeutic effects of TR21 and TR22 in lymphangiogenesis andre-establishment of the lymphatic circulatory system in the rat hindlimb. Effectiveness of TR21 or TR22 treatment is measured by swellingvolume of the affected limb, quantification of the amount of lymphaticvasculature, total blood plasma protein, and histopathology. Acutelymphedema is observed for 7-10 days. Perhaps more importantly, thechronic progress of the edema is followed for up to 3-4 weeks. [0693]Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately 350g are dosedwith Pentobarbital. Subsequently, the right legs are shaved from knee tohip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood isdrawn for serum total protein testing. Circumference and volumetricmeasurements are made prior to injecting dye into paws after marking 2measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). Theintradermal dorsum of both right and left paws are injected with 0.05 mlof 1% Evan's Blue. Circumference and volumetric measurements are thenmade following injection of dye into paws. [0694] Using the knee jointas a landmark, a mid-leg inguinal incision is made circumferentiallyallowing the femoral vessels to be located. Forceps and hemostats areused to dissect and separate the skin flaps. After locating the femoralvessels, the lymphatic vessel that runs along side and underneath thevessel(s) is located. The main lymphatic vessels in this area are thenelectrically coagulated or suture ligated. [06951 Using a microscope,muscles in back of the leg (near the semitendinosis and adductors) arebluntly dissected. The popliteal lymph node is then located. [0696] The2 proximal and 2 distal lymphatic vessels and distal blood supply of thepopliteal node are then and ligated by suturing. The popliteal lymphnode, and any accompanying adipose tissue, is then removed by cuttingconnective tissues. [0697] Care is taken to control any mild bleedingresulting from this procedure. After lymphatics are occluded, the skinflaps are sealed by using liquid skin (Vetbond) (AJ Buck). The separatedskin edges are sealed to the underlying muscle tissue while leaving agap of 0.5 cm around the leg. Skin also may be anchored by suturing tounderlying muscle when necessary. [0698] To avoid infection, animals arehoused individually with mesh (no bedding). Recovering animals arechecked daily through the optimal edematous peak, which typicallyoccurred by day 5-7. The plateau edematous peak are then observed. Toevaluate the intensity of the lymphedema, the circumference and volumesof 2 designated places on each paw before operation and daily for 7 daysare measured. The effect of plasma proteins on lymphedema is determinedand whether protein analysis is a useful testing perimeter is alsoinvestigated. The weights of both control and edematous limbs areevaluated at 2 places. Analysis is performed in a blind manner. [0699]Circumference Measurements: Under brief gas anesthetic to prevent limbmovement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople then those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs. [0700] Volumetric Measurements: On the dayof surgery, animals are anesthetized with Pentobarbital and are testedprior to surgery. For daily volumetrics animals are under briefhalothane anesthetic (rapid immobilization and quick recovery), bothlegs are shaved and equally marked using waterproof marker on legs. Legsare first dipped in water, then dipped into instrument to each markedlevel then measured by Buxco edema software(ChenjVictor). Data isrecorded by one person, while the other is dipping the limb to markedarea. [0701] Blood-plasma protein measurements: Blood is drawn, spun,and serum separated prior to surgery and then at conclusion for totalprotein and Ca2+comparison. [0702] Limb Weight Comparison: After drawingblood, the animal is prepared for tissue collection. The limbs wereamputated using a quillitine, then both experimental and control legswere cut at the ligature and weighed. A second weighing is done as thetibio- cacaneal joint was disarticulated and the foot was weighed.[0703] Histological Preparations: The transverse muscle located behindthe knee (popliteal) area is dissected and arranged in a metal mold,filled with freezeGel, dipped into cold methylbutane, placed intolabeled sample bags at - 80EC until sectioning. Upon sectioning, themuscle was observed under fluorescent microscopy for lymphatics. Otherimmuno/histological methods are currently being evaluated. [07041 Thestudies described in this example test the activity in TR21 and TR22protein. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 and TR22polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21 and TR22. Example 38 TR21-Fc and TR22-Fc inhbition of B cellproliferation in vitro in a co-stimulatory assay [0705] A TR21 orTR22-Fc polypeptide is prepared that consists of a soluble form of TR21or TR22 linked to the Fc portion of a human IgGI immunogloulin molecule.The ability of this protein to alter the proliferative response of humanB cells is assessed in a standard co-stimulatory assay. Briefly, humantonsillar B cells are purified by magnetic bead (MACS) depletion ofCD3-positive cells. The resulting cell population is greater than 95% Bcells as assessed by expression of CD19 and CD20 staining. Variousdilutions of rHuNeutrokine-alpha (Intematioanl Application PublicationNo. WO 98/18921) or the control protein rHuIL2 are placed intoindividual wells of a 96-well plate to which was added 10⁵ B cellssuspended in culture medium (RPMI 1640 containing 10% FBS, 5 X 10- ⁵M2ME, IOOU/ml penicillin, lOug/ml streptomycin, and 10-5 dilution offormalin-fixed Staphylococcus aureus Cowan I (SAC), also known asPansorbin (Pan)) in a total volume of 150ul. TR21 or TR22-Fc is thenadded at various concentrations. Plates are then placed in the incubator(37° C. 5% CO₂, 95% humidity) for three days. Proliferation isquantitated by a 20h pulse (I Ci/well) of ³H-thymidine (6.7 Ci/mM)beginning 72h post factor addition. The positive and negative controlsare IL2 and medium respectively. 10706] It will be clear that theinvention may be practiced otherwise than as particularly described inthe foregoing description and examples. Numerous modifications andvariations of the present invention are possible in light of the aboveteachings and, therefore, are within the scope of the appended claims.[07071 The entire disclosure of each document cited (including patents,patent applications, journal articles, abstracts, laboratory manuals,books, or other disclosures) in the Background of the Invention,Detailed Description, and Examples is hereby incorporated herein byreference. Further, the hard copy of the sequence listing submittedherewith and the corresponding computer readable form are bothincorporated herein by reference in their entireties. [0708] CertainTR21 and TR22 polynucleotides and polypeptides of the present invention,including antibodies, were disclosed in U.S. non provisional applicationnumber 09/910,562, filed July 23, 2001, and provisional applicationnumbers 60/220,116, filed July 24, 2000, and 60/221,143, filed July 27,2000, the specifications of which are each herein incorporated byreference in their entirety.

1. An isolated nucleic acid molecule comprising a polynucleotide havinga nucleotide sequence at least 95% identical to a sequence selected fromthe group consisting of: (a) a nucleotide sequence encoding apolypeptide comprising amino acids 1 to 271 in Figures IA-B; (b) anucleotide sequence encoding a polypeptide comprising amino acids fromabout 30 to about 271 in FIGS. 1A-B; (c) a nucleotide sequence encodinga polypeptide comprising amino acids Asp-30 to Glu-57 in FIGS. 1A-B; (d)a nucleotide sequence encoding amino acids 1 to 194 of FIG. 2; (e) anucleotide sequence encoding a polypeptide having the amino acidsequence encoded by the cDNA clone contained in clone HCFMV39 (ATCCDeposit Number 97974 or 209080) or HMUCLOl (ATCC Deposit Number PTA-2259); (f) a nucleotide sequence encoding the mature TR21 or TR22polypeptide having the amino acid sequence encoded by the cDNA clonecontained in clone HCFMV39 or HMUCLO1, respectively; (g) a nucleotidesequence encoding the extracellular domain of the TR21 or TR22polypeptide having the amino acid sequence encoded by the cDNA clonecontained in clone HCFMV39 or HMUCLO, respectively; (h) a nucleotidesequence encoding the transmembrane domain of the TR21 or TR22polypeptide having the amino acid sequence encoded by the cDNA clonecontained in clone HCFMV39 or HMUCLO, respectively; (i) a nucleotidesequence encoding the intracellular domain of the TR21 or TR22polypeptide having the amino acid sequence encoded by the cDNA clonecontained in clone HCFMV39 or HMUCLO1, respectively; () a nucleotidesequence encoding the TR21 or TR22 receptor extracellular andintracellular domains with all or part of the transmembrane domaindeleted; and (k) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i) or(j).
 2. The nucleic acid molecule of claim 1, wherein saidpolynucleotide comprises the nucleotide sequence in FIG.
 1. 3. Thenucleic acid molecule of claim 1, wherein said polynucleotide comprisesthe nucleotide sequence in FIG.
 2. 4. The nucleic acid molecule of claim1, wherein said polynucleotide has the complete nucleotide sequence ofthe cDNA clone contained in HCFMV39.
 5. The nucleic acid molecule ofclaim 1, wherein said polynucleotide has the complete nucleotidesequence of the cDNA clone contained in HMUCLOI.
 6. An isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a polynucleotide having anucleotide sequence identical to a nucleotide sequence in (0) of claim1, wherein said polynucleotide does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues.
 7. Anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of a TR21 receptorhaving an amino acid sequence in FIGS. 1A-B or encoded by clone HCFMV39.8. An isolated nucleic acid molecule comprising a polynucleotide whichencodes the amino acid sequence of an epitope-bearing portion of a TR22receptor having an amino acid sequence in FIG. 2 or encoded by cloneHMUCLO
 1. 9. An isolated nucleic acid molecule comprising encoding apolypeptide having an amino acid sequence at least 95% identical to asequence selected from the group consisting of: (a) the amino acidsequence of Figures IA-B; (b) the amino acid sequence encoded by thecDNA of clone HCFMV39; (c) the amino acid sequence of FIG. 2; and (d)the amino acid sequence encoded by the cDNA of clone HMUCLO
 1. 10. Anisolated polypeptide encoded by the nucleic acid molecule of claim 9.11. An isolated polypeptide comprising an antigenic epitope contained ina polypeptide having an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence of Figures IA-B; (b) theamino acid sequence encoded by the cDNA of clone HCFMV39; (c) the aminoacid sequence of FIG. 2; and (d) the amino acid sequence encoded by thecDNA of clone HMUCLO1.
 12. An isolated antibody that binds specificallyto the polypeptide of claim
 10. 13. An isolated antibody that bindsspecifically to the polypeptide of claim
 11. 14. A method of treating animmune disorder comprising administering an effective amount of thepolypeptide as claimed in claim 10, or an agonist thereof to a patientin need thereof.
 15. A method of treating an immune disorder comprisingadministering an effective amount of the antibody as claimed in claim12.
 16. An isolated nucleic acid molecule comprising a polynucleotideencoding a polypeptide wherein, except for one to ten conservative aminoacid substitutions, said polypeptide has an amino acid sequence selectedfrom the group consisting of: (a) the amino acid sequence of FiguresIA-B; (b) the amino acid sequence encoded by the cDNA of clone HCFMV39;(c) the amino acid sequence of FIG. 2; and (d) the amino acid sequenceencoded by the cDNA of clone HMUCLOI.
 17. An isolated polypeptidewherein, except for one to ten conservative amino acid substitutions,said polypeptide has a sequence selected from the group consisting of:(a) the amino acid sequence of FIGS. 1A-B; (b) the amino acid sequenceencoded by the cDNA of clone HCFMV39; (c) the amino acid sequence ofFIG. 2; and (d) the amino acid sequence encoded by the cDNA of cloneHMUCLO1.
 18. The antibody of claim 12 that is an scFv fragment.
 19. Theantibody of claim 12 that is an Fab fragment.
 20. A method of inhibitingproliferation of a cell expressing TR21 or TR22, comprising contactingthe cell with an antagonistic antibody or antibody fragment, or otherantagonist of TR21 or TR22.
 21. A method of enhancing proliferation of acell expressing TR21 or TR22, comprising contacting the cell with anagonistic antibody or antibody fragment, or other agonist of TR21 orTR22.